TRNG AI HOC A LAT

F7G

ENZYME

GIAO TRNH

GS.TS. MAI XUAN LNG

2005

Enzyme

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MUC LUC
MUC LUC ............................................................................................................- 1 M AU ..........................................................................................................- 3 I. BAN CHAT PROTEIN CUA ENZYME. ......................................................- 4 II. DANH PHAP VA PHAN LOAI ENZYME..................................................- 6 III. ONG HOC CUA CAC PHAN NG ENZYME. ......................................- 8 IV. NHNG TNH CHAT AC TRNG CUA XUC TAC SINH HOC ........- 12 1. Enzyme the hien tnh ac hieu cao oi vi c chat cua chung..............- 12 2. Xuc tac enzyme dan en s hnh thanh mot phc he trung gian gia enzyme va c chat. .....................................................................................- 12 3. Trung tam cua enzyme tng tac ac hieu vi c chat c goi la trung tam hoat ong. ...................................................................................................- 12 4. Enzyme lam giam nang lng hoat hoa can thiet cho mot phan ng......- 13 5. Mot so enzyme tham gia ieu hoa toc o phan ng..............................- 14 6. Mot so enzyme la multienzyme hay phc he a chc nang. ..................- 15 7. ong hoc cua phan ng enzyme hai c chat.........................................- 15 8. Anh hng cua pH ................................................................................- 16 9. Anh hng cua nhiet o. .......................................................................- 18 V. C CHE ENZYME....................................................................................- 19 1. c che canh tranh (competitive inhibition)............................................- 19 2. c che khong canh tranh kieu th I (noncompetitive inhibition)...........- 22 3. c che khong canh tranh kieu th II (uncompetitive inhibition). ..........- 23 VI. CAC CHAT C CHE TRAO OI CHAT- ANTIMETABOLITE ...........- 24 VII. HE THONG MULTIENZYM VA VAI TRO CUA ENZYME IEU HOA. .. 26 VIII. HE THONG CASCADE - BIEN OI ONG HOA TR. ...............- 29 IX. HOAT HOA ENZYME. ...........................................................................- 31 X. TNG TAC PROTEIN - PROTEIN......................................................- 32 XI. TNH AC HIEU CUA ENZYME OI VI C CHAT.........................- 33 XII. C CHE TANG TOC O CAC PHAN NG HOA HOC NH ENZYME... 36 1. Tang toc o phan ng va tnh ac hieu c chat.....................................- 36 2. S phu hp cam ng va xuc tac enzyme................................................- 38 3. C che tiep can. .....................................................................................- 38 4. Gay mat on nh (Destabilization).........................................................- 40 5. Xuc tac acid-base phoi hp....................................................................- 41 XIII. ISOENZYME.........................................................................................- 46 XIV. CAC NHOM ENZYME.........................................................................- 47 1. Enzyme oxy hoa - kh. ...........................................................................- 47 2. Transferase. ............................................................................................- 53 3. Hydrolase................................................................................................- 55 4. Liase. ......................................................................................................- 57 5. Isomerase................................................................................................- 58 6. Ligase (synthetase).................................................................................- 59 -

GS.TS. Mai Xuan Lng

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Enzyme

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XV. TACH CHIET VA TINH CHE ENZYME. .............................................- 60 XVI. S DUNG ENZYME TRONG CONG NGHE SINH HOC. ..................- 65 XVII. ENZYME CO NH. ...........................................................................- 68 1.Y ngha cua enzyme co nh....................................................................- 68 2. Cac phng phap ieu che enzyme co nh............................................- 68 3. Mot so ac tnh cua enzyme co nh.......................................................- 74 4. ng dung cua enzyme co nh................................................................- 75 -

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Enzyme

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M AU
Enzyme la cac chat xuc tac cua cac he thong sinh hoc. Chung co kha nang xuc tac ac biet, thng la manh hn nhieu so vi cac chat xuc tac tong hp. Tac dung xuc tac cua chung mang tnh ac hieu cao oi vi c chat, lam tang ang ke toc o cac phan ng hoa hoc xay ra trong moi trng nc ieu kien nhiet o va pH em du. Enzyme la mot trong cac cha khoa e hieu biet qua trnh hoat ong song cua te bao. Hoat ong trong nhng trat t co tnh to chc cao, chung xuc tac hang tram phan ng theo trat t xac nh trong cac con ng trao oi chat ma nh o cac chat dinh dng b phan huy, nang lng hoa hoc c lu gi va bien oi, cac ai phan t sinh hoc c tao ra t cac chat tien than n gian. Mot so enzyme tham gia trong qua trnh trao oi chat la nhng enzyme ieu hoa, chu trach nhiem oi vi cac tn hieu trao oi chat khac nhau bang cach thay oi hoat tnh xuc tac cua chung mot cach thch hp. Thong qua hoat ong cua cac enzyme ieu hoa cac he thong enzyme phoi hp chat che vi nhau e tao ra moi quan he hai hoa gia cac hoat tnh trao oi chat can thiet cho viec duy tr s song. Nghien cu enzyme con co y ngha thc tien rat quan trong. oi vi mot so benh, ac biet la cac roi loan mang tnh di truyen, co the la do thieu hay mat han mot hoac mot so enzyme trong cac mo. Cac ieu kien khong bnh thng cung co the xuat hien do hoat tnh d tha cua mot so enzyme ac hieu. Xac nh hoat tnh cua mot so enzyme xac nh trong huyet tng, hong cau hoac trong cac mo la rat quan trong trong viec chan oan benh. Enzyme a tr thanh cac cong cu thc te quan trong khong nh.ng trong y hoc ma ca trong cong nghe hoa hoc, trong che bien thc an va trong nong nghiep. Enzyme co vai tro tham ch trong hoat ong hang ngay cua gia nh, v du nh trong viec lau chui cho ban hoac trong cong viec che bien thc an.

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Khoa Sinh hoc

Enzyme I. BAN CHAT PROTEIN CUA ENZYME.

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Phan ln lch s hoa sinh hoc la lch s nghien cu enzyme. Cac chat xuc tac sinh hoc lan au tien c phat hien va mo ta vao nhng nam 1800 trong cac nghien cu ve tieu hoa tht bang cac chat tiet cua da day va s bien oi tinh bot thanh ng bi nc bot va bi cac dch chiet thc vat khac nhau. Trong nhng nam 1850 Louis Pasteur ket luan rang qua trnh len men ng thanh ru bi nam men c xuc tac bi ferment. Ong cho rang nhng men nay, ma ve sau c goi la enzyme, la nhng chat khong tach ri khoi cau truc cua te bao nam men song, mot quan iem ton tai trong nhieu nam. Cho en nam 1897 Eduard Buchner a xac nh rang cac dch chiet nam men co the len men ng thanh ru ngay ca khi chung c tach khoi cau truc cua te bao nam men. Phat hien nay a thuc ay cac nha sinh hoa tm cach tach chiet nhieu enzyme khac nhau va nghien cu hoat tnh xuc tac cua chung. Cong trnh tach chiet va tinh che urease cua James Sumner nam 1926 a thuc ay cac nghien cu au tien tnh chat cua cac enzyme ac hieu. Sumner a phat hien c rang cac tinh the urease c cau tao hoan toan t protein va t o ong cho rang tat ca enzyme la protein. Y tng nay qua cac v du khac a tiep tuc c tranh cai them nhieu nam sau o. Ch en nhng nam cuoi cua thap ky 1930, sau khi John Northrop va cac cong tac vien cua ong ket tinh c pepsin va trypsin va cung xac nh c chung cung la protein th quan niem cua Sumner ve enzyme mi c cong nhan rong rai. Ngay nay hoa sinh hoc a xac nh c rang tat ca enzyme la protein. Hoat tnh xuc tac cua chung phu thuoc vao tnh nguyen ven cua cau truc nguyen thuy cua protein. Neu mot enzyme b bien tnh hoac b phan ly thanh cac phan di n v th hoat tnh xuc tac cua no thng b mat. Khi mot enzyme b phan giai thanh aminoacid th hoat tnh xuc tac cua no hoan toan khong con. Nh vay, cau truc bac mot, bac hai, bac ba va bac bon cua protein enzyme la nhng yeu to rat quan trong oi vi hoat tnh xuc tac cua chung. Enzyme, cung nh cac protein khac, co trong lng phan t t khoang 12.000 en hn 1.000.000. Mot so enzyme khong can cac nhom hoa hoc khong phai aminoacid cho hoat tnh xuc tac cua mnh. Mot so khac can co cac nhom bo sung goi la cofactor (bang 1) Nhng cofactor nay co the la mot hoac mot so ion kim loai nh Fe2+, Mg2+, Mn2+,hoac Zn2+ hoac mot phan t hu c hay hu c cha lim loai phc tap c goi la coenzyme (bang 2). Mot so enzyme oi hoi ca coenzyme va mot vai ion kim loai cho hoat tnh cua mnh. Mot coenzyme hoac ion kim loai lien ket cong hoa tr vi protein enzyme c goi la nhom them hay nhom prosthetic. Mot enzyme tron ven

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co hoat tnh xuc tac cung vi coenzyme va (hoac) ion kim loai hp lai c goi la holoenzyme. Phan protein cua loai enzyme nay c goi la apoenzyme hay apoprotein. Coenzyme hoat ong nh vat mang cac nhom chc ac hieu. Nhieu vitamin va cac chat hu c vi ham lng nho co trong thc an la cac chat tien than cua coenzyme. Bang 1. Mot so enzyme co cha hoac can cac nguyen to vo c e lam cofactor. Cofactor Fe hoac Fe3+ Cu2+ Zn2+ Mg2+ Mn2+ K+ Ni2+ Mo Se
2+

Enzyme Cytochrome Oxydase Catalase, Peroxydase Cytochrome Oxydase Carbonic Anhydrase, Alcohol Dehydrogenase Hexokinase, Glucoso-6-phosphatase, Pyruvate Kinase Arginase, Ribonucleotide reductase Pyruvate kinase Urease Dinitrogenase Glutathione peroxidase

Bang 2. Mot so coenzyme lam vat trung chuyen cac nguyen t hoac cac nhom nguyen t ac hieu.. Chat tien than trong thc Nhom c van COENZYME an cua ong vat co vu chuyen Thiamine pyrophosphate Flavine adenine dinucleotide Nicotinamide dinuclotide Coenzyme A Pyridoxal phosphate 5Deoxyadenosylcobalamine (Coenzyme B12) Aldehyde ien t ien t Nhom acyl Nhom amine Cac nguyen t H va nhom alkyl Thiamine (Vitamine B1) Riboflavine (Vitamine B2) Nicotinic acid (Niacin) Acid pantothenic Pyridoxine (Vitamine B6) Vitamine B12

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Enzyme Biocytin Tetrahydrofolate Acid lipoic

CO2 Nhom mot carbon ien t va nhom acyl

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Biotin
Folate Khong can co trong thc an

II. DANH PHAP VA PHAN LOAI ENZYME

Ten goi cua enzyme thng la ten goi cua c chat hay cua kieu phan ng ma no xuc tac cong vi uoi ase, v du urease,, hydrolase v.v... Ngoai ra con co nhng ten goi truyen thong theo thoi quen, khong cho thay ban chat hoa hoc cua phan ng do enzyme xuc tac, v du pepsin, trypsin ... ca hai kieu goi ten neu tren eu thieu chnh xac. e khac phuc tnh trang o, Hoi Hoa sinh hoc quoc te e ngh s dung mot he thong danh phap va phan loai tren c s ban chat cua phan ng c xuc tac. Theo he thong nay toan bo enzyme c goi ten theo ban chat cua phan ng c xuc tac va ban chat cua cac chat cho, chat nhan trong phan ng va c chia thanh 6 nhom ln; moi nhom ln nay lai c chia thanh nhieu phan nhom; moi phan nhom nay lai c chia thanh nhieu phan nhom nho hn, trong o bao gom nhng enzyme co c chat tac dung giong nhau. Moi nhom, moi phan nhom va moi enzyme c ky hieu bang mot ma so ac trng gom tng ng mot, hai, ba hoac bon con so cach nhau bang cac dau cham. Ten goi cua 6 nhom enzyme va cac phan nhom quan trong c gii thieu trong bang 3 cung vi ban chat cua cac phan ng c xuc tac. Cac phan nhom nho hn thuoc moi phan nhom trong bang 3 c ky hieu bang nhng ma so gom 2 hoac 3 con so. V du phan nhom th nhat cua enzyme nhom 1 (ky hieu la phan nhom 1.1) co ba phan nhom nho au tien la 1.1.1, 1.1.2 va 1.1.3 ac trng cho cac trng hp ma chat nhan ien t la NAD, NADP va cytochrome. Ma so cua moi enzyme gom 4 con so, v du: 1.1.1.29 Glycerophosphate dehydrogenase; 2.7.1.1 Hexokinase 3.2.1.20 - Glucosidase; 4.1.1.1 Pyruvate decarboxylase; 5.3.1.1 Triosophosphate isomerase; 6.3.1.2 Glutamin synthetase Bang 3. Danh muc ma so cua 6 nhom enzyme va cac phan nhom chnh cua chung Nhom Phan nhom Phan ng c xuc tac

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Enzyme
1. Oxydoreductase 1.1 1.2 1.3 1.4 1.5 1.6 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 3.1 3.2 3.3 3.4 3.5 3.6 4.1 4.2 4.3 5.1 5.2 5.3 5.4 6.1 6.2 6.3 6.4

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2. Transferase

Hydrogen hoa va dehydrogen hoa =CHOH =C=O CH=CH CHNH2 =CHNH NADH, NADPH Van chuyen cac nhom chc Cac goc 1 carbon Nhom aldehyde hoac cetone Acyl Lien ket glycoside Nhom methylalkyl hoac aryl Nhom cha nit Nhom cha phosphore Nhom cha lu huynh Cac phan ng thuy phan Ester Glycoside Eter Peptide Cac lien ket C-N khac Cac anhydrit acid Tao lien ket oi =C=C= =C=O =C=N ong phan hoa Rasemase va epimerase Xis-trans-isomerase Oxy hoa noi phan t Transferase noi phan t Tao ra lien ket nh ATP C=O CS =C=N CC

3. Hydrolase

4. Liase

5. Isomerase

6. Ligase

Khi ten he thong cua enzyme qua dai hoac s dung khong thuan tien, ngi ta co the dung ten goi thong dung cua chung, v du ten he thong cua enzyme xuc tac phan ng ATP + D-Glucose ADP + D-Glucoso-6-phosphate la ATP:glucose phosphotransferase; ten goi nay cho thay enzyme xuc tac s van chuyen nhom phosphate t ATP en glucose. Ma so cua enzyme la 2.7.1.1: so 2 cho biet enzyme thuoc nhom th 2; con so 7 cho biet enzyme thuoc phan nhom phosphotransferase; so 1 tiep theo cho biet chat nhan

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nhom phosphate la nho OH; So 1 cuoi cung cho biet chat nhan nhom phosphate la D-glucose. Khi ten he thong cua enzyme qua dai co the dung ten thong dung cua no, trong trng hp nay co the goi ten enzyme la hesokinase

III. ONG HOC CUA CAC PHAN NG ENZYME.

Bat ky phan ng hoa hoc nao, v du phan ng A P, s d xay ra c la nh mot phan nang lng trong so cac phan t A cha nang lng ln hn so phan t con lai, lam cho chung ton tai trang thai hoat ong. trang thai nay de dang pha v mot lien ket hoa hoc hoac tao ra mot lien ket mi e lam xuat hien san pham P. Nang lng can e chuyen toan bo so phan t cua mot mol vat chat ieu kien nhat nh sang trang thai kch ong c goi la nang lng hoat hoa. Nang lng nay can thiet e chuyen cac phan t tham gia phan ng sang mot trang thai trung gian giau nang lng tng ng vi nh cua hang rao hoat hoa (hnh 1). Toc o cua phan ng t le vi nong o cua phan t trang thai trung gian nay. Nang lng hoat hoa c o bang nang lng can thiet e chuyen cac phan t len trang thai hoat ong. Chat xuc tac lam giam nang lng hoat hoa von can e phan ng co the xay ra t phat. Bang 4 cho biet nang lng hoat hoa oi vi mot so phan ng. Phan ng phan huy peroxide hydro oi hoi 18.000 KCal/mol nhng se giam xuong con 11.700 khi co platin xuc tac va con giam thap hn na khi chat xuc tac la enzyme catalase. Ro rang, catalase co hieu qua hn nhieu so vi chat xuc tac vo c oi vi phan ng nay. Tren thc te catalase co hieu qua en mc ch can mot gia tr nang lng hoat hoa rat nho cho phan ng. V vay ma phan giai H2O2 bang catalase xay ra hau nh ngay tc khac vi toc o nhanh nhat trong so cac phan ng enzyme a biet. Bang 4. con cho thay cac enzyme khac cung giam nang lng hoat hoa xuong mc thap hn ang ke so vi cac chat xuc tac vo c. V ly do o ma cac phan ng enzyme co the xay ra vi toc o cao ieu kien nhiet o sinh ly. Bang 4 . Nang lng hoat hoa oi vi cac phan ng xuc tac bang enzyme va bang cac chat xuc tac khac. Phan ng Phan giai peroxide hydro Chat xuc tac khong platin catalase ion hydro ion hydroxyl lipase tuyen tuy Ea (Kcal/mol) 18.000 11.700 < 2.000 16.800 10.200 4.500

Thuy phan ethyl butyrate

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Khoa Sinh hoc

Enzyme
Thuy phan casein Thuy phan saccharose Thuy phan -methylglucoside ion hydro trypsin ion hydro invertase nam men ion hydro - glucosidase 20.600 12.000

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25.000 8.000 -10.000 32.600 12.200

Khi tang nhiet o nang lng chuyen ong nhiet cua phan t tang len, lam cho so phan t co kha nang at trang thai trung gian tang len. V the khi tang nhiet o len 10o, toc o cua phu hoa hoc tang len khoang hai lan (Q10 = 2). Khac vi tac dung cua nhiet o, chat xuc tac lam tang toc o cua phan ng bang cach lam giam nang lng hoat hoa. S ket hp gia chat phan ng va chat xuc tac lam xuat hien trang thai trung gian mi vi mc nang lng hoat hoa thap hn. Khi san pham hnh thanh, chat xuc tac Hnh 1. Bien thien nang lng t do lai c giai phong trong cac phan ng hoa hoc. trang thai t do. Cac phan ng enzyme cung tuan theo nhng nguyen tac chung cua ong hoc cac phan ng hoa hoc. Tuy nhien, chung con co nhng ac iem rieng. Mot trong nhng ac iem o la hien tng bao hoa c chat. nong o c chat thap toc o cua phan ng enzyme t le thuan vi nong o c chat. Nhng neu tiep tuc tang nong o c chat th toc o phan ng tang cham dan, va khi nong o c chat at mot gia tr nao o, toc o cua phan ng khong tang na. Trong nhng ieu kien o nong o enzyme la yeu to quyet nh toc o phan ng. Mac du hien tng bao hoa c chat ac trng cho moi enzyme, nhng gia tr cu the cua nong o c chat la gia tr ac trng. Thong qua nghien cu van e nay ong Leonor Michaelis (1857-1949) va ba Maud Menten (18791960) a e xuat vao nam 1913 mot phng trnh dien ta toc o cac phan

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Enzyme

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ng enzyme va neu len mot so ly thuyet chung ve ong hoc cua qua trnh nay. Thuyet nay ve sau a c Briggs va Haldans phat trien them. Cac tac gia tren nhan thay rang trong cac phan ng enzyme trc tien enzyme E tao ra phc he ES vi c chat S. Sau o ES se c phan giai thanh san pham P va enzyme E t do. Theo nh luat khoi lng, qua trnh o co the c mo ta nh sau: k3 k1 E+S ES E+P k4 k2 trong o k1 la hang so toc o phan ng hnh thanh ES t E va S; k2 la hang so toc o phan ng phan giai ES thanh E va S; k3 la hang so toc o phan ng phan giai ES thanh E va P; k4 la hang so toc o phan ng hnh thanh ES t E va P. trang thai can bang toc o hnh thanh ES bang toc o phan giai phc he nay: k1[E][S] k2[ES] = k3[ES] k4[E][P]. Bien oi phng trnh nay, ta co: [ES](k2+k3) = [E](k4[P] + k1[S]) [ES] k4[P] + k1[S] k4[P] k1[S] k2+k3 k2+k3 [E] k2 + k3 Do cac giai oan au cua phan ng gia tr cua [P] vo cung nho nen co the gian lc phng trnh tren nh sau: [ES] k1[S] [E] k 2 + k3 at [E]t la ham lng enzyme tong so va Km = k2+k3 / k1, ta co: [E] [ES] [E]t -[ES] [ES] [E] [ES] Km [S]

Toc o ban au v cua phan ng enzyme t le thuan vi ham lng enzyme hoat ong, hay ES], nen ta co the viet: v = k3[ES] Neu nong o c chat rat ln, lam cho hau het enzyme trong he thong eu ton tai trang thai ES, th toc o phan ng enzyme se at gia tr toi a V, va toc o toi a o se bang: V = k3[E]t Do o: [E] V Km [ES] v [S]

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Enzyme
Nhan hai ve cho [S] va bien oi phng trnh, ta co: v = V[S] Km + [S]

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ay chnh la phng trnh Michaelis-Menten va Km c goi la hang so Michaelis. Y ngha thc tien cua hang so Michaelis la cho no chnh la gia tr cua nong o c chat khi toc o phan ng bang toc o toi a. Thay V va v bang cac con so tng ng 1 va 0,5 vao phng trnh tren, ta se thay ro ieu o. Nh vay, Km c o bang n v nong o, tc mol/l. Hang so Michaelis la mot hang so rat quan trong. No xac nh ai lc cua enzyme vi c chat. Km cang nho th ai lc nay cang ln, toc o phan ng cang cao v toc o toi a V at gia tr nong o c chat cang thap. Tren c s phng trnh Michaelis-Menten, bang cach xay dng ng bieu dien s phu thuoc cua v vao [S] va bang o th o xac nh toc o toi a V ta co the tm thay gia tr cua [S], o v = V/2, tc gia tr cua Km (hnh 2). Tuy nhien, bang cach nay kho xac nh v mot cach chnh xac. e khac phuc nhc iem o, ngi ta s dung ng bieu dien Linewear-Burk. Hai tac gia

Hnh 2. ng bieu dien phng trnh Michaelis-Menten

nay bien oi phng trnh Michaelis-Menten thanh dang: 1/v = Km/V x 1/[S] + 1/V u iem cua phng trnh nay la cho gia cac ai lng 1/v va 1/[S] co moi lien he t le thuan (hnh 3). Qua ng bieu dien nay ta co the thay rang tang ABO = Km/V va BO = 1/Km. Phng trnh nay con cho phep tm hieu nhieu kha canh quan

Hnh 3. ng bieu dien phng trnh Linewear-

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Khoa Sinh hoc

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trong lien quan en tac dung cua cac chat c che hoat tnh cua enzyme.

IV. NHNG TNH CHAT AC TRNG CUA XUC TAC SINH HOC
1. Enzyme the hien tnh ac hieu cao oi vi c chat cua chung. Mot so enzyme ch xuc tac mot phan ng chuyen hoa mot c chat. V du fumarase ch xuc tac phan ng chuyen hoa gia fumarate va malate: OH COOOOC - CH2 - C - COO- CH = CH + H2O H OOC L-Malate Fumarate Ca maleat - ong phan dang cis cua fumarat - va D-malat eu khong the la c chat cua fumarase. Cac Enzyme khac co tnh ac hieu rong hn. V du moi enzyme thuy phan protein trong bang 2.4 co tnh ac hieu vi cac lien ket peptide von hnh thanh bi cac aminoacid khac nhau, ong thi cung the hien tnh ac hieu lap the, ch thuy phan cac lien ket peptide hnh thanh bi cac L- ch khong phai cac D-aminoacid. Tuy nhien cung co nhng enzyme co tnh ac hieu rong hn, v du mot so enzyme thuy phan protein cung co the thuy phan ca cac lien ket ester va tyoester. 2. Xuc tac enzyme dan en s hnh thanh mot phc he trung gian gia enzyme va c chat. S hnh thanh cac phc he enzyme-c chat nh nhng chat trung gian trong cac phan ng enzyme a c phat hien bang nhng bien phap khac nhau, bao gom phan tch ong hoc, s dung cac thuoc th ac hieu oi vi goc R e tao ra cac bien oi hoa hoc, c che enzyme bang cac hp chat ac hieu tng tac vi trung tam hoat ong, phat hien quang pho hap thu ac hieu khi enzyme tac dung vi c chat, dung tia X phat hien cau truc tinh the cua enzyme ket hp vi cac hp chat tng t ve mat cau truc vi c chat... 3. Trung tam cua enzyme tng tac ac hieu vi c chat c goi la trung tam hoat ong. Hnh dang cua mot so enzyme cho phep mot so nhom R xac nh trong chuoi polypeptide c nam canh nhau mot cach rat ac hieu e tao ra cac trung tam hoat ong. Cau truc khong gian tai trung tam hoat ong khong ch xac nh hp chat nao co the phu hp ve mat lap the oi vi trung tam

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Khoa Sinh hoc

Enzyme

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ma con quy nh ban chat cua cac bien co tiep theo e lam cho c chat bien hoa thanh san pham. S ket hp cua c chat vi trung tam hoat ong co the c thc hien thong qua s hnh thanh cac lien ket khong ong hoa tr ac hieu va trong mot vai trng hp, ca lien ket ong hoa tr. Khi lien ket tai trung tam, c chat c at gan sat vi cac nhom ac hieu cua enzyme, gay ra s mat on nh cua mot so lien ket nhat nh trong c chat, do o lam cho chung tr nen hoat ong hn ve mat hoa hoc. 4. Enzyme lam giam nang lng hoat hoa can thiet cho mot phan ng. nhiet o khong oi, mot tap oan cac phan t co mot ong nang phan bo gia cac phan t nh mo ta mot cach khai quat trong hnh 4a. nhiet o T1 tap oan cac phan t khong co u nang lng e thc hien mot phan ng hoa hoc ac hieu nao o, nhng neu nhiet o c nang len en T2 th s phan bo nang lng thay oi theo. Tai T2 bay gi co u nang lng e nang so va cham gia cac phan t, lam cho mot phan ng hoa hoc co the xay ra. Nh vay, khi nhiet o c nang len t T1 en T2 viec tang toc o phan ng chu yeu la ket qua cua viec tang so phan t c hoat hoa, tc bo phan co c nang lng can thiet cho s hoat hoa . Hnh 3c cho thay bc tranh n gian ve mat nang lng cua mot tap oan cac phan t trong qua trnh phan ng A B. Khi phan ng xay ra, co u so phan t vi mc nang lng can thiet e tr nen hoat ong va tham gia trang thai trung chuyen, tai o chung phan hoa thanh san pham. Nang lng can e at trang thai trung chuyen, hay trang thai hoat hoa la nang lng hoat hoa (Ea ). e mot phan ng co the xay ra, mc nang lng cua cac chat phan ng phai ln hn cua san pham. Tong bien thien nang lng cua phan ng la mc henh lech gia cac mc nang lng cua A va cua B. Enzyme, cung nh moi chat xuc tac, lam tang toc o cua cac phan ng hoa hoc bang cach lam giam nang lng hoat hoa cua phan ng ac hieu nh ta co the thay trong cac hnh 4b va 4c.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 14 -

Hnh 4. (a) Phan bo ong nang cua mot tap oan phan t nhiet o T1 va T2 cao hn. Mui ten ch mc nang lng toi thieu can thiet e cac phan t tham gia phan ng. Tai T1 phan ng khong xay ra, nhng T2 phan ng c thc hien. (b) ong nang cua tap oan cac phan t c chat nhiet o T1 cac mui ten ch cac mc nang lng can thiet e xay ra phan ng khi vang mat va khi co mat enzyme. Can chu y rang khi khong co enzyme th phan ng khong xay ra, con khi co mat enzyme phan ng co the c thc hien ma khong can thay oi nhiet o. (c) Bien thien nang lng cua phan ng khong co enzyme xuc tac va co enzyme xuc tac A B. Trong phan ng khong co enzyme xuc tac, mc nang lng cua A can c nang len u e hoat hoa cac phan t cua A va a chung len trang thai trung chuyen A,B* , tai o no co the phan ng vi B. Nang lng can e mang cac phan t len trang thai trung chuyen c goi la nang lng hoat hoa Ea . Mc chenh lech gia cac mc nang lng cua A va cua A.B* c ch bang so 1. Trong phan ng co xuc tac Ea can e tao ra cac phc he hoat ong ES c ch bang so 2 thap hn nhieu so vi so 1 cua qua trnh khong xuc tac. S chenh lech gia cac mc nang lng gia A va B (so 3) la nh nhau trong ca 2 phan ng co xuc tac cung nh khong co xuc tac. 5. Mot so enzyme tham gia ieu hoa toc o phan ng. a so c the khong thay oi toc o cua cac phan ng trao oi chat khi nhiet o bien oi. V vay cac phan ng xuc tac can lam cho qua trnh xay ra u nhanh nhiet o cua c the. Hn na, neu cac phan ng sinh hoc xay ra khong co xuc tac th khong the kiem tra c toc o cua chung. Hang loat cac c che ieu hoa c s dung e ieu hoa qua trnh trao oi chat. Mot so hoat ong mc o cua ban than enzyme. Mot chat co tac dung lam tang hoac giam toc o cua mot phan ng enzyme, bang cach tac ong trc tiep len enzyme xuc tac c goi la chat hieu ng (effector). Cac chat hieu ng the hien tac dung cua chung bang cach lam thay oi cau truc

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 15 -

cua enzyme sao cho ch gay anh hng en toc o phan ng. C che ieu hoa cua enzyme se c xem xet sau. 6. Mot so enzyme la multienzyme hay phc he a chc nang. Cac phc he multienzyme co trong lng phan t ln thng cha t ba enzyme khac nhau tr len ket hp chat che vi nhau bang cach tng tac khong ong hoa tr va ch co the phan ly trong cac ieu kien lam pha v cac lien ket khong ong hoa tr. Moi enzyme trong phc he xuc tac mot phan ng rieng biet nhng cung vi cac enzyme khac cua phc he xuc tac mot phan ng tong the duy nhat. Pyruvat dehydrogenase la mot v du ve phc he multi- enzyme.Cac enzyme khac nam trong cac phc he a chc nang (multifuntional complex), trong o hai hay nhieu hn cac enzyme rieng biet cha trong nhng khu vc rieng cua mot chuoi polypeptide duy nhat. Moi khu vc rieng o xuc tac mot bc cua mot phan ng tong the duy nhat do phc he a chc nang xuc tac. Synthetase acid beo la mot v du cho loai phc he nay. 7. ong hoc cua phan ng enzyme hai c chat. a so enzyme co nhieu hn mot c chat va xuc tac cac phan ng co dang A + B C + D. Cac phan ng nay dan en s hnh thanh cac phc he enzyme - c chat giong nh cac phan ng mot c chat va ong hoc cua chung co the c dung e thu nhan Km oi vi moi c chat c o bang phan tch o th (theo cac phng trnh 11 - 13) cua toc o ban au khi thay oi nong o cua c chat nay va gi nguyen nong o bao hoa cua c chat kia. Phng trnh ong hoc cua phan ng hai c chat tng t nh phng trnh toc o Michelis - Menten cho phep hieu mot cach sau sac c che chung cua cac phan ng loai nay va xac nh ga tr cua cac hang so ong hoc nh Km va Vmax. Nhng phng trnh nay rat phc tap nen khong the e cap en ay. Tuy nhien, se rat bo ch neu xem xet cac c che c ban cua cac phan ng hai c chat bao gom hai c che khac nhau la c che thay the kep (double displacement mechanism) va c che lien tuc (sequental mechanism). Trong c che thay the kep oi vi phan ng A + B C + D, mot c chat (A) gan vi enzyme e tao ra phc he EA. E va A sau o phan ng e tao ra phc he mi FC va sau o mot san pham (C) c giai phong e tao ra phc he trung gian enzyme - c chat F khac vi E. San pham trung gian F sau o phan ng vi c chat th hai (B) e tao ra phc he enzyme - c chat FB. Phc he nay se tao ra san pham th hai D va khoi phuc enzyme E. C che nay co the c mo ta mot cach khai quat dang s o sau ay: A C B D

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme
E + A (EA F C) F + B (FB FD) E

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A, B, C, D la chat phan ng va san pham, E la enzyme, F la dang trung gian cua enzyme. C che nay con c goi la c che ping-pong. Phan ng chuyen amin-hoa bang enzyme glutamic-aspartic aminotransferase la mot v du ve c che nay. C che lien tuc co hai loai: loai trat t va loai tuy tien. Ngc vi c che ping-pong, trong c che lien tuc tat ca cac c chat co the ket hp e tao ra mot phc he ba thanh phan trc khi san pham hnh thanh. Cac phan ng loai tat t co the c mo ta dang s o sau ay: A B C D

E + A EA + B (EAB ECD) ED E Cac phan ng xuc tac bi phosphofructokinase va glycealdehyde-3phosphate dehydrogenase la nhng v du ve kieu phan ng trat t cua c che lien tuc. Trong cac trng hp khac, E mang cac trung tam ket hp oi vi ca A va B va toc o phan ng khong b anh hng bi A hoac B c gan trc vao trung tam danh cho chung. V vay, c che nay c goi la c che tuy tien. Neu cung khong co mot trat t "thch hp" cho s giai phong cac san pham C va D sau khi cac phc he ba thanh phan EAB bien thanh ECD, th c che tuy tien co the c mo ta nh sau: A B C D

ED E EB A B

EA (EAB ECD) C D EC

V du ve c che tuy tien la cac phan ng xuc tac bi cac enzyme UDP-ga- lactose:N-Acetylgalactosamine galactosyl transferase va creatine kinase. Cac c che ong hoc phc tap hn loi cuon ba va tham ch bon c chat tham gia phan ng. Chung co the thuoc c che lien tuc hoac c che pingpong hoac la s phoi hp cua ca hai c che. 8. Anh hng cua pH pH anh hng ang ke en toc o phan ng. Nhieu phan ng enzyme co toc o nhanh nhat mot ga tr pH c goi la pH toi thch, trong khi cac phan ng enzyme khac co toc o nh nhau trong mot pham vi cac ga tr pH

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 17 -

xac nh. Bang 5 cho thay pH toi thch oi vi mot so enzyme. Mot so yeu to co anh hng en pH toi thch bao gom cac goc acid tai trung tam hoat ong . Neu mot enzyme oi hoi mot nhom acide protein hoa cho hoat ong cua mnh th enzyme o co the co hoat tnh cao nhat tai cac ga tr pH thap hn pK cua nhom o, ngc lai, neu can dang phan ly cua mot acide th hoat tnh cao nhat se the hien tai cac gia tr pH cao hn pK cua nhom o. Thong thng co hai nhom acide phan ly tr len tham gia tai trung tam hoat ong va ng cong hoat tnh theo pH se phan anh s phu thuoc vao moi nhom. tren thc te, nghien cu anh hng cua pH oi vi toc o phan ng co the giup xac nh cac nhom acid tai trung tam hoat ong, mac du cung can ca nhng thong tin khac.

Bang 5. pH toi thch cua mot so enzyme thuy phan. Enzyme Pepsin C chat Albumin trng casein Hemoglobin Benzyloxycarboxylglutamyltyrosine -Metylglucoside Maltose Urea Protein Tinh bot Tinh bot Cac chat khac nhau 2-Glycerophosphate 2-Glycerophosphate Arginine pHopt 1,5 1,8 2,2 4,0 5,4 7,0 6,4-6,9 7,8 6,7-7,2 7,8 7,5 9-10 4,5-5,0 9,5-9-9

-Glucosidase Urease Trypsin -Amylase tuyen tuy -Amylase mam lua mach Carboxypeptidase Phosphatase kiem huyet tng Phosphatase acid huyet tng Arginase

Mot so c chat la nhng acide yeu hoac cha nhng thanh phan ion va pH toi thch co the phan anh thc trang la enzyme can dang ion hay dang khong phai ion. Them vao o toc o phan ng thng giam rat nhanh khi pH giam thap hay qua cao co the cho thay enzyme b bien tnh hay b phan ly mot cofactor quan trong nao o.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme
9. Anh hng cua nhiet o.

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Hang so can bang cua moi phan ng hoa hoc cung nh toc o cua phan ng phu thuoc rat nhieu vao nhiet o. Cac phan ng enzyme cung khong phai la ngoai le. Anh hung cua nhiet o len hang so can bang cua mot phan ng hoa hoc c mo ta bang phng trnh van't Hoff: H 2,3 logK = C - RT Trong o H la nhiet lng cua phan ng tnh bang calo/mol, R la hang so kh bang 1,98 cal/mol/oC va T la nhiet o tuyet oi. C la mot hang so hp nhat (integration constant). T phng trnh nay co the thay o th cua logK oi vi 1/T la mot ng thang. o nghieng cua ng thang nay la H / 2,3R. Anh hng cua nhiet o len toc o cua mot phan ng c mo ta bang phng trnh Arrhenius: Ea 2,3 logK = B - RT Phng trnh nay cung co dang nh phng trnh (19) nhng mo ta quan he cua hang so toc o k cua phan ng vi T, R, Ea (nang lng hoat hoa tnh bang calo/mol) va hang so B bieu hien nh lng tan so va cham va yeu cau nh hng ac hieu gia cac phan t va cham. Hnh 5 . Anh hng cua nhiet o len hang so toc o k2 cua cac phan ng thuy phan benzyloxycarbonyl-glycylphenylalanin (CGP) va benzyloxycarbonylglycyltryptophan (CGT) bang carboxypep-tidase tinh the. Cac so lieu ghi nhan bang o th dang log k2 oi vi gia tr nghch ao cua nhiet o tuyet oi (1/T). Nang lng hoat hoa bieu kien Ea bang 9.900 cal/mol oi vi CGT va 9.600 oi vi CGP. Hnh 5 cho thay anh hng cua nhiet o len hang so toc o k2 cua phan ng thuy phan hai c chat bi carboxypeptidase. ay la mot o th Arrhenius ien hnh oi vi mot phan ng enzyme von cho thay logk2 thay oi ty le thuan vi 1/T trong pham vi t 5 en 25oC va cho thay Ea co the c xac nh t o nghieng cua ng thang. oi vi cac phan ng enzyme, toc o phan ng tang theo nhiet o cho en khi at c toc o toi a, nhng nhiet o cao hn mc toi a se lam giam toc o phan ng do enzyme b bien tnh.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme V. C CHE ENZYME

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Toc o cua cac phan ng enzyme b giam bi tac dung cua cac chat c che ac hieu, tc nhng chat ket hp vi enzyme va ngan can enzyme ket hp mot cach bnh thng vi c chat. Tnh oc cua nhieu chat nh HCN va H2S la do tac dung cua chung nh mot chat c che enzyme. Nhieu loai thuoc cung co tac dung c che cac enzyme ac hieu. Do o, hieu biet cac chat c che enzyme la mot ieu rat quan trong e hieu tac dung cua thuoc va cac chat oc. Hn na thong tin ve ban than enzyme cung thu c bang cach nghien cu cac chat c che enzyme. Co ba kieu c che thuan nghch mang cac ac iem ong hoc khac nhau. o la mot kieu c che canh tranh (competitive inhibition) va hai kieu c che khong canh tranh (noncompetitive va uncompetitive inhibition). 1. c che canh tranh (competitive inhibition). Cac chat c che canh tranh co the ket hp thuan nghch vi trung tam hoat ong cua enzyme va canh tranh vi c chat e gianh lay trung tam hoat ong. Khi trung tam hoat ong a b chat c che chiem gi, no khong the ket hp vi c chat. S ket hp cua chat c che canh tranh I vi enzyme E co the c mo ta giong nh s ket hp gia enzyme va c chat, mac du chat c che khong chuyen hoa thanh san pham: E + I EI Hang so pha.n ly Ki cua phc he EI la: [E][I] Ki = [EI] V s hnh thanh EI phu thuoc vao [I] cung nh s hnh thanh ES phu thuoc vao [S] nen toc o thc te cua phan ng c che canh tranh hoan toan phu thuoc vao nong o tng oi cua S va I tai mot nong o xac nh cua E. Mot trong nhng v du ien hnh nhat cua c che canh tranh la anh hng cua acid malonic oi vi enzyme succinate dehydrogenase xuc tac phan ng sau ay khi co mot chat nhan hydro A thch hp : COOCH2 CH2 + A
-

HCCOOOOCCH + AH2

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme
COOSuccinate Chat nhan Fumarate

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Chat nhan dang kh

Nhieu hp chat vi cau truc giong vi acid succinic la nhng chat c che canh tranh cua loai enzyme dehydrogenase nay, bao gom: COOH COOH COOH CH2 C=O CH2 CH2 COOH Oxalate COOH CH2 COOH Malonate CH2 CH2 CH2 COOH Glutarate CH2 COOH COOH CH2

Phenylpropionate Oxaloacetate

Manh nhat trong so cac chat c che nay la acid malonic. Khi ty le [I]/[S] = 1/50, enzyme a b c che 50%. Tang nong o cua c chat khi [I] khong oi, se lam giam mc o c che, va ngc lai, giam nong o c chat se lam tang mc o c che. Neu acid succinic va acid malonic gan vi cac trung tam khac nhau cua enzyme th khong the giai thch c v sao chung canh tranh vi nhau. V chung canh tranh nen co the ket luan rang chung ket hp vi enzyme tai cung mot cho, o la trung tam hoat ong. Cau truc cua moi chat c che canh tranh giong vi c chat tai mot so kha canh nao o. Cac chat c che canh tranh co the c nhan biet bang ac iem ong hoc qua hieu ng cua nong o chat c che oi vi quan he gia v va [S] nh minh hoa bang o th cua phng trnh Lineweaver-Burk. Tac dung cua c che canh tranh tuan theo phng trnh sau ay vi s tham gia cua Ki - hang so phan ly cua EI: 1 Km [I] 1 1 = 1 + + Ki [S] Vmax v Vmax ac iem cua c che competitive la co cung iem cat truc tung (1/Vmax ) nh phan ng khong c che nhng o nghieng th khac vi phan ng khong c che bi gia tr 1 + [I]/Ki. o th trong hnh 6a cho thay ro khi nong o cua S cao th phan ng t b c che, ngc lai khi nong o cua S giam th mc o c che tang len cung vi cac gia tr [I]/[S] va Ki .

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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Hnh 6. o th ao ngc kep mo ta cac kieu c che phan ng enzyme: (a): c che competivive, (b): c che noncompetitive, (c): c che uncompetitive. Km va Vmax c xac nh t o doc va cac iem cat cua cac phan ng khong c che, con Ki - t o doc va/hoac cho cat cua cac phan ng b c che.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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2. c che khong canh tranh kieu th I (noncompetitive inhibition). Trong trng hp nay khong co moi quan he gia mc o c che vi nong o c chat. c che ch phu thuoc vao nong o cua chat c che. Trai ngc vi c che canh tranh, ngi ta cho rang s hnh thanh EI xay ra tai ni khong phai e enzyme gan vi c chat. E+I va ES + I ESI EI

Ca EI va ESI eu la nhng phc he khong hoat ong. Co hai hang so phan ly: [E][I] EI Ki = [EI] va [ESI] la: KiESI = [ES][I]

Phng trnh ao ngc kep oi vi kieu c che khong canh tranh nay 1 Km = V Vmax [I] 1 1 [I] 1 + + 1 + [Ki ] [S] Vmax Ki

Phng trnh c che canh tranh kieu th nhat tren ay va ng bieu dien cua no trnh bay trong hnh 6b. Ca o nghieng va iem cat eu khac vi trng hp khong c che bi gia tr 1+[I]/Ki. Cac chat c che noncompetitive khong phan ng tai trung tam hoat ong ma tai mot ni nao o tren phan t enzyme dan en s bien oi ang ke hnh dang cua enzyme e ngan can trung tam hoat ong ket hp mot cach bnh thng vi c chat.V du ve c che noncompetitive la cac kim loai nang nh Ag+, Hg2+, Pb2+ von tng tac thuan nghch vi cac nhom thyol cua enzyme hoac cac yeu to tao phc chelat ma hieu ng c che la do chung ket hp vi cac ion kim loai ma rat can e the hien hoat tnh xuc tac. Nhieu hp chat ket hp khong thuan nghch vi enzyme va tao ra cac dan xuat ong hoa tr tai trung tam hoat ong hay tai mot bo phan khac cua phan t khong tham gia trc tiep trong tng tac enzyme-c chat. ay khong phai la c che noncompetitive vi y ngha chat che v chung c che enzyme mot cach khong thuan nghch. V du papain cha mot nhom thyol duy nhat tai trung tam hoat ong, no phan ng rat nhanh chong vi iodoacetate e tao ra nhom S-carboxylmethylcysteine. Mc o c che papain bi chat c che nay ty le thuan vi mc o S-carboxymethyl-hoa. Iodacetate cung c che

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 23 -

mot so enzyme co cha nhom thyol khong phai tai trung tam hoat ong ma lam suy yeu hoat tnh xuc tac do lam bien oi cau truc cua phan t enzyme. 3. c che khong canh tranh kieu th II (uncompetitive inhibition). Kieu c che nay xay ra khi mot chat c che ch ket hp thuan nghch vi phc he ES e tao ra ESI ma sau o khong the tao ra san pham (cac chat c che noncompetitive co the ket hp ca vi enzyme t do va vi phc he ES), nh vay: Ki [ES][I] Va phng trnh ao ngc kep se la 1 V 1 1 [I] Km = x + 1 + [S] Vmax Ki Vmax [ESI] =

o th cua phng trnh nay (hnh 6c) cho thay kieu c che nay dan en s thay oi ac trng iem cat truc tung nhng khong thay oi o nghieng cua o th so vi trng hp khong c che. Cung nh c che noncompetitive, kieu c che uncompetitive khong the ao ngc bang cach tang nong o c chat. Kieu c che nay thng tm thay trong cac phan ng enzyme vi hai c chat tr len.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 24 -

VI. CAC CHAT C CHE TRAO OI CHAT- ANTIMETABOLITE

Chat trao oi (metabolite) la nhng hp chat hnh thanh trong cac qua trnh trao oi chat bnh thng cua c the, con cac chat chong trao oi (antimetabolite) la nhng chat co cau truc giong vi mot chat trao oi nao o va, khi co mat trong c the, chung c che viec s dung chat trao oi o. Cac chat antimetabolite c che sinh trng va oi khi co the giet chet c the, mac du tac dung c che sinh trng co the c khac phuc bang cach cung cap chat trao oi can thiet. Antimetabolite a c s dung e xac nh cac chat trao oi quan trong, hay cac yeu to sinh trng, ac biet oi vi vi sinh vat. Tuy nhien, s quan tam hien nay oi vi antimetabolite thng vi muc ch s dung chung nh nhng chat c che sinh trng oi vi cac vi sinh vat gay benh va te bao ung th. Nh vay, neu mot antimetabolite c phat hien co kha nang c che sinh trng cua vi sinh vat gay benh ma khong anh hng ang ke en trao oi chat cua sinh vat chu th no co the c xem xet e s dung nh mot chat khang sinh (antibiotic). Tng t, neu no c che sinh trng cua te bao ung th vi mc o ln hn la c che sinh trng cua cac mo chu th no co the tr thanh mot loai thuoc tr ung th (antitumor agent) co hieu qua. Nhieu antimetabolite la nhng chat c che cac enzyme ac hieu. Do o, hieu biet nhng enzyme nay se giup tao ra cac antimetabolite, mac du nhieu chat khang sinh va khang ung th a c phat hien theo phng phap kinh nghiem. e giai thch cach tac dung ac hieu cua antimetabolite len enzyme, ta se xem xet ay c che tac dung cua mot nhom chat khang sinh goi la "thuoc sulfa" Viec nghien cu antimetabolit bat au c ac biet chu y khi phat hien c rang s c che sinh trng cua vi khuan bi sulfanilamide b ngan can mang tnh canh tranh bi mot yeu to sinh trng la acid p-aminobenzoid. S giong nhau ve mat cau truc cua hai chat la rat ro, va hien tng nay cung giong nh trng hp c che canh tranh cua enzyme. NH2 NH2

SO2NH2

COOH Acid p-aminobenzoic Sulfanilamide

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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Thc vay, acid p-aminobenzoic co the khac phuc mang tnh canh tranh tac dung c che cua tat ca cac sulfonamide co cau truc NH2-C6H4SO2NHR, v du sylfaguanidine, sulfathiazol, sulfapyridine va sulfadiazine. Nhng c the von can acid p-aminobenzoic (PABA) cho sinh trng s dung no e tong hp acid folic. Sinh trng cua nhng c the nay b c che bi cac loai sulfonamide va s c che nay co the b ao ngc bi PABA. Nhng c the von can acid folic e sinh trng va khong the s dung PABA th khong b c che bi sulfonamide. Nh vay, sulfonamide c che (cac) phan ng enzyme dan en dan en tong hp acid folic t acid paminobenzoic va cac chat tien than khac. Co le s dung cac loai sulfonamide e chong nhiem khuan ngi mot cach co hieu qua la do c the ngi can acid folic nhng khong tong hp acid nay t PABA. Nh vay, sulfonamide ngan can phan ng trao oi can thiet oi vi vi khuan ma khong anh hng en trao oi chat cua c the chu von khong tong hp acid folic t PABA.

Mot so chat oi khang cua acid folic cung a c s dung mc o nhat nh e ieu tr benh bach cau va cac benh ung th khac. V du acid 4amino-pteroylglutamic (aminopterin) c che sinh trng cua mot so loai ung th.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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VII. HE THONG MULTIENZYM VA VAI TRO CUA ENZYME IEU HOA.

Trong te bao nhieu enzyme hoat ong ong thi, trong o san pham cua phan ng trc la c chat cua phan ng sau. V du qua trnh bien oi glucose thanh acid lactic c thc hien bang mot trat t cac phan ng tao thanh qua trnh glycolis. Trong cac he thong multienzyme nay san pham cua phan ng trc la c chat cua phan ng sau. Trong moi he thong multienzyme thong thng co mot enzyme chu trach nhiem chi phoi toc o cua toan bo he thong, c goi la enzyme ieu hoa. Tuy thuoc vao mc o phc tap, nhng he thong multienzyme (hay a enzyme) nay c chia lam 3 loai (hnh 7): 1/ Cac enzyme ca biet hoa tan trong te bao chat va hoat ong oc lap nhau. Cac phan t c chat co kch thc nho, de khuech tan, co the tm thay nhanh chong con ng t enzyme nay sang enzyme khac (1); 2/ cac enzyme cua he thong ket hp nhau thanh mot phc he hoat ong phoi hp nhau. o la trng hp he enzyme tong hp acid beo cua nam men. He thong nay gom 7 enzyme ket hp chat che vi nhau. Neu tach ri nhau, tat ca eu b mat hoat tnh; 3/ Co mc o to chc cao nhat la nhng he thong enzyme lien ket vi cac cau truc tren phan t cua te bao. o la trng hp oi vi he enzyme cua chuoi van chuyen ien t trong ti the. Moi enzyme ca biet gan vao lp mang trong cua ti the, va lam nhiem vu xuc tac, va ong vai tro nh mot yeu to cau truc cua mang. Trong nhng he thong multienzyme nay toc o cua mot phan ng nao o thng xac nh toc o cua toan bo

Hnh 7. Cac kieu to chc cua he thong

he thong, trong o yeu to han che co the la nong o enzyme hoac nong o c chat. Hoat tnh cua enzyme ieu hoa c ieu chnh thong qua thong qua cac kieu phan t tn hieu khac nhau von la cac chat trao oi phan t nho hoac cac cofactor. Co hai loai enzyme ieu hoa tham gia trong cac con ng trao doi chat khac nhau. Loai th nhat la cac enzyme allosteric hay d lap the. Chung hoat ong thong qua cac lien ket khong ong hoa tr vi cac chat trao

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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oi lam nhiem vu ieu hoa goi la modulator.Ch allosteric tieng Hy lap co ngha la cau nh khac. Enzyme allosteric la nhng enzyme co cau hnh khac khi lien ket vi cac modulator. Loai enzyme ieu hoa th hai bao gom nhng enzyme c ieu hoa bang cach lien ket ong hoa tr thuan nghch vi modulator. Ca hai loai enzyme ieu hoa nay c cau tao t cac phan di n v, va trong mot so trng hp co cac trung tam ieu hoa va trung tam hoat ong nam tren cac phan di n vi khac nhau. t nhat con hai c che khac e ieu hoa hoat tnh cua enzyme.. Mot so enzyme c kch thch hoac b c che bi cac protein kiem tra rieng biet khi nhng protein nay gan vi enzyme va anh hng en hoat tnh cua chung. Mot so enzyme khac c hoat hoa bang tac ong thuy phan khong thuan nhch ac biet oi vi phan t enzyme. Cac v du quan trong ve hai c che nay co the tm thay trong cac qua trnh sinh ly nh tieu hoa, nghen mau, hoat ong cua hormone ... Trong mot so he thong multienzyme enzyme ieu hoa b c che ac hieu bi cac san pham cuoi cung cua chuoi phan ng khi ham lng cua san pham o vt qua nhu cau cua te bao. Khi phan ng cua enzyme ieu hoa thap cac enzyme tiep theo cung se giam toc o hoat ong do c chat cua chung b giam, do o lam giam so lng san pham cua toan bo he thong multienzyme. Kieu ieu hoa nay c goi la ieu hoa theo nguyen tac lien he ngc.

Hnh8. c che Ltreonine desaminase bi i l i th

V du ien hnh ve kieu ieu hoa nay la ieu hoa hoat ong cua he thong multienzyme, trong o L-threonine chuyen hoa thanh L-isoleucine bao gom 5 phan ng (hnh 8). Enzyme E1 (threonine desaminase) b c che bi san pham cuoi cung L-isileucine, mac du no khong phai la chat canh tranh vi L-threonine. Tat ca nhng aminoacid giong no eu khong gay hieu ng nay. Ro rang la khi trong he thong tch luy qua nhieu L-isoleucine, vt qua mc cho phep nao o, th enzyme au tien cua he thong se b c che. Chat trao oi gay tac dung c che enzyme ieu hoa c goi la effector am tnh, hay modulator am tnh. Cung co trng hp enzyme ieu hoa nam iem phan nhanh cua cac day phan ng. Enzyme ieu hoa co the la n tr,

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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neu ch chu tac dung cua mot effector, hoac a tr neu chu tac dung cua t hai effector tr len. Cac effector co the la cac san pham cuoi cung cua nhng trat t phan ng lien quan nhau. Nh ac iem nay mot so he thong multienzyme co the co chung mot he thong ieu hoa. Enzyme ieu hoa co the chu tac dung cua effector dng tnh. Thong thng, effector dng tnh la c chat cua chnh enzyme ieu hoa. Co nhng trng hp enzyme ieu hoa chu tac ong ong thi cua mot hoac mot so effector am tnh va mot hoac mot so effector dng tnh, trong o moi effector ket hp vi be mat enzyme tai mot v tr rat ac trng. Phan ln enzyme ieu hoa trong ieu kien te bao la nhng enzyme xuc tac cac phan ng khong thuan nghch. S ton tai cua enzyme ieu hoa la mot trong nhng the hien cua nguyen tac tiet kiem toi a cua te bao.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme VIII. HE THONG CASCADE - BIEN OI ONG HOA TR.

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ieu hoa hoat tnh enzyme con co the c thc hien bang cach bien oi enzyme theo chu trnh kn gia dang bien oi ong hoa tr va dang khong bien oi. S bien oi co tnh chu ky nay c thc hien nh cac enzyme bien hoa (converter enzymes), chung cung vi cac enzyme b bien oi va khong b bien oi va cac chat hieu ng cua chung tao thanh mot he thong cascade. Mot enzyme b bien oi ong hoa tr co the tr nen hoat ong hn so vi dang khong bien oi, hoac hai dang co the co cach phan ng khac nhau vi cac chat gay hieu ng. He thong cascade n gian nhat la he thong n chu ky (monocycle) va hoat ong cua no c mo ta trong hnh 9 oi vi mot enzyme gia thuyet von bien oi ong hoa tr bang cach phosphoryl-hoa. Qua trnh bien oi c bat au khi enzyme bien hoa Ei dang khong hoat ong c hoat hoa nh mot chat cam ng d lap the ac hieu e1 thanh dang hoat ong Ea. Enzyme cha b bien oi I0 sau o b phosphoryl hoa bi Ea trong mot phan ng phu thuoc ATP e bien thanh enzyme b bien oi Im va ADP. Qua trnh nay co tnh thuan nghch v mot enzyme bien hoa th hai Ra dang hoat ong, von c hnh thanh nh s hoat hoa dang khong hoat ong Ri va chat hieu ng d lap the e2 , se diphoshporyl hoa Im e tao lai I0 va Pi . Cac he thong cascade n chu ky khac nhau co the ket hp vi nhau e tao ra cac he thong bi-, tri-, va multicascade ma vi o nhay cao oi vi nong o cua chat hieu ng co the ieu hoa mot cach tinh vi dong c chat va san pham xuyen qua mang li cac con ng trao oi chat. Cac he thong casade co kha nang ieu hoa toc o phan ng bang nhieu cach: 1/ chung cung cap tn hieu khuyech ai, tc la ch mot lng nho enzyme bien hoa (Ea hay Ra) tao ra c mot lng ln enzyme bien oi (Im) hoac enzyme khong bien oi. 2/ Chung co the ieu chnh mc o toi a ma Im co the at c vi so lng bao hoa cua e1. 3/ Chung co the ieu chnh mc o nhay cam cua s bien oi oi vi nhng thay oi nong o cua chat cam ng. 4/ Chung c s dung nh nhng he thong hp nhat (integration systems) cam nhan c nhng bien oi rat nho cua nong o noi bao cua cac chat trao oi va ieu chnh nong o cua Io va Im cho thch hp. 5/ Chung la nhng he thong linh hoat co kha nang thc hien cac kieu ap ng khacnhau oi vi cac kch thch thch d lap the. 6/ Chung la bo khuech ai toc o ap ng mc mili-giay oi vi nhng bien oi cua nong o cac chat trao oi trong te bao.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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Hnh 9. S o hoat ong cua he thong cascade n chu ky cho phep phosphoryl hoa enzyme khong bien oi Io thanh dang phosphorylhoa Im di anh hng cua cac enzyme bien hoa Ea va R a von c hnh thanh nh tng tac vi cac chat cam ng e1 va e2 vi cac enzyme bien hoa khong hoat ong tng ng Ei va Ri . Ea va Ra xuc tac cac phan ng phosphoryl-hoa va dephosphoryl hoa Io va Im . Phosphoryl hoa cac nhom hydroxyl cua serine, threonine hoac thyrosine trong cac enzyme xay ra trong nhieu he thong cascade; hn 20 enzyme c biet la nhng enzyme chu phosphoryl hoa thuan nghch bi he thong tng t nh a mo ta trong hnh 9. Moi he thong nay s dung ATP nham cung cap nang lng e duy tr lng enzyme bien oi tng ho thch hp von can e ieu hoa toc o phan ng. Nhng qua trnh rat khac nhau nh sinh tong hp va phan giai glycogen, sinh tong hp cholesterol, chuyen hoa aminoacid eu c ieu hoa theo kieu nay. Mot so he thong cascade c ieu hoa bi cac kieu bien oi ong hoa tr khac vi phosphorryl-hoa. V du glutamine synthetase cua E. coli hoat ong trong mot he thong cascade keo theo nucleotide hoa cac enzyme hoac cac protein ieu hoa chung bang phan ng vi ATP hoac UTP.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme IX. HOAT HOA ENZYME.

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Nhieu enzyme e the hien hoat tnh cua mnh, can phai co s ho tr cua cac yeu to khac nhau, trong o moi enzyme c hoat hoa bang mot con ng nhat nh. Bon kieu hoat hoa khac nhau c mo ta trong hnh 10. Mot so enzyme, v du pepsinogen, trypsinogen ... c hoat hoa bang cach cat bo mot oan oligopeptide khoi phan t proenzyme (1); Hnh 4. Cac kieu hoat hoa enzyme

Hnh 10. Cac kieu hoat hoa enzyme Mot so enzyme khac c hoat hoa bang cach hnh thanh cau disulfide, v du ribonuclease (2), hoac bang cach tao phc vi ion kim loai (3). Kieu hoat hoa th t ac trng cho cac enzyme d lap the, c thc hien bang cach thay oi cau hnh khong gian cua enzyme nh mot effector dng tnh ac hieu (4).

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme X. TNG TAC PROTEIN - PROTEIN.

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Cac enzyme d lap the thc hien viec kiem tra cac phan ng enzyme bang cach tng tac phoi hp gia cac phan di n v. Cac enzyme khac, v du proteinkinase ton tai dang khong hoat ong do cac tng tac protein protein gia cac phan di n v cua chung. Proteinkinase cua c van ton tai dang mot holoenzyme khong hoat ong vi cau truc di n v R2C2, trong o R la phan di n v ieu hoa, con C la phan di n v xuc tac. Kinase la mot enzyme bien hoa va c hoat hoa bi cAMP nh sau: R2C2 + 2cAMP R2(cAMP) + 2C S ket hp cua cAMP vi R a giai phong C e co the xuc tac phan ng phosphorryl-hoa hang loat cac enzyme trong cac he thong cascade. Mot v du khac cua tng tac protein - protein trong ieu hoa hoat tnh enzyme trng hp oi vi lactose synthetase - enzyme co nhiem vu tong hp lactose trong tuyen sa cua ong vat co vu. Lactose synthetase xuc tac phan ng: UDP-Galactose + Glucose UDP. Galactosyl- -1,4-glucose (Lactose) +

Enzyme synthetase nay can enzyme UDP-galactose:N-acetylglucosamine galactosyl transferase von co mat trong nhieu mo khong phai tuyen sa va tham gia vao viec tong hp cac nhom prostetic co ban chat oligosacchaside cua mot so glycoprotein, v du: UDP-Gal + GlcNAc...Protein Gal- -1-GlcNAc...Protein + UDP. Trong te bao tuyen sa cua ong vat co vu galactosyl transferase lien ket tren mang tng tac vi mot protein hoa tan la -lactalbumin e tao ra lactose synthase, enzyme xuc tac tong hp lactose. Trong tng tac lactalbumin vi galactosyl-transferase tnh ac hieu c chat cua transferase c bien oi sao cho glucose tr thanh chat nhan c chat. Glucose la mot chat nhan rat yeu (Km = 1 2M) oi vi transferase nay, song, khi co mat -lactalbumin, no tr thanh mot chat nhan rat tot. (Km = 10 13M).

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme XI. TNH AC HIEU CUA ENZYME OI VI C CHAT.

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Enzyme khac mot cach ro ret vi cac chat xuc tac hoa hoc khac tnh ac hieu c chat va hieu qua xuc tac. Phan ln enzyme ch co mot so t cac c chat t nhien e c bien hoa thanh san pham n gian vi hieu qua rat cao. Cau truc oc ao cua trung tam hoat ong cua enzyme quy nh tnh ac hieu nay va khong ch cho phep ket hp mot cach thuan li vi c chat ac hieu ma con loai tr kha nang lien ket khong thch hp cua nhieu chat khong phai la c chat cua enzyme. Mc o ac hieu cao nay c duy tr cung vi toc o phan ng nhanh gap 106 - 1012 lan so vi cac phan ng t phat khong xuc tac. Nhieu enzyme co tnh ac hieu tuyet oi oi vi mot c chat duy nhat. o la trng hp cua suxinate dehydrogenase va fumarase. Cac enzyme khac co tnh ac hieu rong hn, v du trypsin thuy phan tat ca cac lien ket peptide, amide va ester hnh thanh vi s tham gia cua lysine hoac arginine. Mac du co the thuy phan cac kieu lien ket khac nhau, trypsin co tnh ac hieu mot cach nghiem ngat vi cac nhom R cua lysine va arginine. V du, cac dan xuat -N-benzoylamide cua homoarginine va ornitine khong phai la c chat, trong khi o cac dan xuat nay cua arginine va lysine lai b thuy phan rat nhanh chong thanh -N-benzoylaminoacid va NH3. S d nh vay la v homoarginine cha mot nhom -CH2 - nhieu hn trong goc R cua no so vi arginine, con ornitine lai cha mot nhom -CH2 - t hn so vi lysine.

C=O NH3+ HN O

C=O NH2+ HN O

H2C - (CH2)3 - CH - C - NH2

H2N - C -NH -(CH2)3 -CH - C - NH2

-Benzoyl-L-lysinamide

-Benzoyl-L-argininamide

C=O C=O NH3+ HN O NH2+ HN O H2C - (CH2)2 - CH - C - NH2 H2N - C -NH -(CH2)4 -CH - C - NH2

-Benzoyl-L-ornitinamide

-Benzoyl-L-homoargininamide

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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Cac nghien cu tinh the cho thay rang tnh ac hieu nay gan lien vi phan ng cua nhom cation cua c chat vi nhom COO- cua acid aspartic tai trung tam hoat ong. S oi cho cua nhom cation do tang hoac giam mot nhom -CH2- a ngan can s lien ket cua c chat va do o khong cho phep enzyme thc hien phan ng deamine-hoa. Cac enzyme khac phan ng vi cac hp chat khac nhau va co tnh ac hieu tng oi rong. Leucine aminopeptidase la mot v du. Enzyme nay thuy phan nhieu amide cua -L-aminoacid va dipeptide vi toc o khac nhau trong cac phan ng co dang nh sau: NH2 O H NH3+ R C - C - NHR + H2O R - C -COO- + H3NR H

trong o R la goc aminoacid con R' la nguyen t hydro (trong amide) hay mot goc aminoacid khac (trong dipeptide). Moi c chat can co mot nhom amine khong b thay the va mot nguyen t hydro tai carbon nam canh lien ket amide hoac peptide nhay cam. Goc aminoacid NH2-tan cung phai co cau hnh L, tr glycine von cung ch rat t khi tham gia trong viec tao ra c chat cho enzyme nay. Tnh ac hieu cua cac enzyme noi tren cho thay rang kch thc, hnh dang va ban chat hoa hoc cua cac nhom tren c chat xac nh toc o ma c chat chu s tac ong cua enzyme. Nhng d kien co c ngay nay cho phep ngh rang trong viec hnh thanh s ket hp mang tnh bo sung gia c chat vi trung tam hoat ong cua enzyme co the co s tham gia cua cac tng tac k nc, tnh ien cung nh lien ket hydro. Trong mot so trng hp cac chat trung gian ong hoa tr cung co the hnh thanh mot cach tam thi trong cac phc he enzyme-c chat. Nh vay, tat ca cac nhom cua c chat c lap at mot cach st sao vao trung tam hoat ong sao cho moi nhom nam mot cach chnh xac ben canh cac nhom bo sung sao cho moi nhom nam mot cach chnh xac ben canh nhom bo sung trong trung tam hoat ong. Muc ch chnh cua chung ta la hieu c bang cach nao kieu lien ket ac hieu nh vay cuoi cung dan en s bien oi hoa hoc i oi vi viec gan c chat tai trung tam hoat ong cua enzyme. Tuy nhien, trc khi xem xet nhng c che nay, can phai tm hieu cac c che cua viec thuc ay nhanh toc o cua cac phan ng enzyme. Co hai c s cau truc quan trong xac nh tnh ac hieu cua enzyme oi vi c chat. o la:

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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1/ C chat can cha kieu lien ket hoa hoc ac trng ma enzyme co the cong pha; 2/ Ben canh yeu to th nhat, c chat con cha mot hoac mot so nhom chc co kha nang ket hp vi enzyme bang cach nao o e nh hng c chat tai trung tam hoat ong, tc trung tam phan ng, cua enzyme. V du ien hnh la trng hp acetylcholine esterase phan giai lien ket ester gia choline va goc acetyl (hnh 11). Kha nang cua enzyme phan giai lien ket ester phu thuoc ca vao s ton tai cua cac goc serine, tyrosine va histidine von trc tiep tham gia qua trnh phan ng, cung nh vao s co mat cua nhom COO- e lien ket tnh ien vi N+cua c chat.

Hnh 11. S phu hp ve cau truc gia enzyme va c chat

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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XII. C CHE TANG TOC O CAC PHAN NG HOA HOC NH ENZYME.

Enzyme lam giam nang lng hoat hoa cua phan ng va bang cach o tang toc o cua phan ng. Kieu tang toc o nay can c giai thch bang li le cua cac phan ng hoa hoc xay ra khi c chat tng tac vi enzyme. Cac c che nay ro rang co quan he vi nhng c che xac nh tnh ac hieu c chat. 1. Tang toc o phan ng va tnh ac hieu c chat. Cac kieu phan ng hoa hoc xay ra trong qua trnh xuc tac cua enzyme cung tng t nh trong cac phan ng hoa hoc hu c. Tuy nhien, kha canh ac hieu cua tac dung enzyme la tnh ac hieu c chat va tnh chat cua nhieu enzyme cho thay rang nang lng lien ket ac hieu cua cac tng tac nhieu thanh phan von xay ra gia cac nhom bo sung trong c chat va trong trung tam hoat ong c s dung e cung cap ong lc cho xuc tac ong gop mot phan quan trong trong viec giam nang lng hoat hoa cua phan ng, va nh vay lam cho toc o cua tat ca cac phan ng enzyme tang len ang ke. ieu nay c bieu th nhieu enzyme, nhng gay an tng nhat la phosphoglucomutase xuc tac phan ng thuan nghch sau ay: -D-Glucoso-1-phosphate -D-Glucoso-6-phosphate

Phan ng nay ch xay ra khi co mat Mg2+ va glucoso-1,6-diphosphate; dang trung gian enzyme phosphoryl-hoa c hnh thanh, trong o nhom phosphate tren goc serine duy nhat cua enzyme trao oi vi cac c chat va vi glucoso-1,6-diphosphate: Enz-OH + Glucozo-1,6-di(P) Enz-O-(P) + Glucoso-1-(P). bi

Bang6. Toc o tng oi cua phosphorryl-hoa mot so chat phosphoglucomutase.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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* Toc o phosphorryl-hoa cua nc c xem la bang 1e so sanh vi toc o phospho-ryl hoa cua cac chat khac. Hang so toc o bac 1 cua phan ng phosphorryl-hoa nc la 3,2x10-8 /s 30oC va pH 7,5. Khi phan ng lap lai, mot trong cac ester phosphate cua glucose co the bien thanh dang kia. Enzyme phosphoryl-hoa ben trong moi trng nc, nhng phan ng vi hang loat cac chat co cau truc chung R-OH theo phan ng Enz-O-(P) + R-OH Enz-OH + R-O-(P) So sanh toc o phosphorryl-hoa cac c chat khac nhau trong bang 6 cho phep phat hien moi quan he ly thu gia s lien ket c chat va hieu qua xuc tac. Chuyen nhom phosphate cua enzyme cho nc (thuy phan enzyme phosphate) xay ra rat cham, vi toc o ch khoang 60 lan phan ng thuy phan enzyme serine phosphate khi khong co enzyme xuc tac. Ngc lai, phosphite va xyloso-1-phosphate ma ca hai eu khong phai la c chat lai tang toc o van chuyen phosphate cho nc tng ng 580 va 1,7x105 lan. ieu nay cho thay rang kha nang phan ng cua nhom phosphoryl cua enzyme c nang len ang ke bi cac chat ma ve mat cau truc tng t vi c chat va co the gan tai trung tam hoat ong; phosphite gan tai trung tam danh cho phosphate, con xylose tai trung tam danh cho ng. Qua bang 6 ta co the thay xylose c phosphorryl-hoa bi phosphoryl-enzyme nhanh hn 7x104 lan so vi phosphoryl-hoa nc, con khi co mat phosphite no c phosphorryl-hoa thanh xyloso-1-phosphate nhanh hn phosphorryl-hoa nc

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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en 2x109 lan va nhanh hn phosphorryl-hoa c chat t nhien la glucoso-1phosphate 15 lan. Cuoi cung, mot so monophosphodiol mach thang, trong o co nhom hydroxyl cach nhom phosphoryl 4 nguyen t carbon cung hoan toan co the b phosphorryl-hoa bi enzyme nay vi toc o khoang 105 -107 lan nhanh hn phosphorryl-hoa nc. Nh vay, viec lien ket tai trung tam phosphate va trung tam ng bi cac hp chat vi cau truc thch hp dan en giam ang ke nang lng hoat hoa cua cac phan ng phosphorryl-hoa bi phospho-glucomutase. Cac so lieu trong bang 6 hoan toan ung ho quan iem cho rang cac lc manh lien quan vi s ket hp cua c chat vi trung tam hoat ong cung c loi cuon vao cac bien co hoa hoc dan en nang cao ang ke toc o bien hoa cua c chat. Vi khai niem nay chung ta co the xem xet cac kieu c che tham gia vao viec ay nhanh toc o cua cac phan ng enzyme va phu thuoc vao nang lng lien ket cua c chat vi enzyme. 2. S phu hp cam ng va xuc tac enzyme. Ngi ta cho rang nhieu enzyme khi khong co mat c chat ton tai dang khong hoat ong va khong phai tat ca cac nhom trong trung tam hoat ong eu nh hng ung trong khong gian e tng tac vi cac nhom bo sung cua c chat. Tuy nhien, s ket hp cua c chat ac hieu se dan en s bien oi hnh dang trong enzyme, trong o cac nhom cua trung tam hoat ong xe dch en cac v tr can thiet e s xuc tac co the c thc hien. Nhng bien oi hnh dang c cam ng bi c chat nh vay c goi la s phu hp cam ng cua tac dung enzyme. No a c minh hoa trong hnh 1.6. Nhieu dan chng ve hieu ng nay a c ghi nhan khi so sanh cau truc enzyme bang phng phap so sanh cau truc tinh the bang tia X trong cac trng hp co mat va vang mat chat c che, v du oi vi carboxypeptidase A va lysozyme. Them vao o, tnh chat cua enzyme trong dung dch cung cho thay nhng khac biet ve hnh dang khi co mat va vang mat c chat. V du, mot so enzyme mat kha nang phan ng vi khang the ac hieu cua chung khi co mac c chat, con mot so enzyme khac th cho thay s khac biet ve hang so lang. Noi chung, ngi ta cong nhan rang s phu hp cam ng co the lam thay oi toc o cua mot so phan ng enzyme nhng tren quy mo tang toc toan bo th co mc o thap hn cac c che khac. 3. C che tiep can. Con ng ro nhat e enzyme nang cao toc o cua mot phan ng hai phan t (bimolecular reaction) la keo hai chat phan ng lai gan nhau trong trung tam hoat ong. Cac phan t phan ng c nh hng mot cach ung an va c tiep can vi nhau lam cho nong o hieu lc tr nen ln hn nhieu so vi trong dung dch loang. Do cac lc lien ket manh va a dang

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 39 -

gia c chat va cau truc cua trung tam hoat ong, enzyme co the lam tang kha nang e hai c chat co the en vi nhau e thc hien phan ng va bien mot cach co hieu qua phan ng hai phan t thanh phan ng mot phan t (noi phan t). Hieu ng nay co nhieu ten goi khac nhau nhng eu vi y ngha la s tiep can. Hieu ng tiep can co the c minh hoa mot cach ro ret nhat bang mo hnh phan ng noi phan t va hieu ng cua cau truc len toc o. Bang 7 gii thieu cau truc cua mot so ester p-bromophenyl cua suxinate va glutarate va toc o tng oi cua cac phan ng thuy phan chung so vi toc o cua phan ng hai phan t thuy phan p-bromophenylacetate vi s xuc tac cua acetate. Moi chat b thuy phan bang tan cong nucleophyl noi phan t cua nhom carbonyl ben canh theo phng trnh phan ng tong quat sau ay.

O C - OR C O O
-

O C -RO C O O +H2O

O C - OH C - OH O

Nhng ester co cau truc cng hn tang kha nang e nhom carboxyl tan cong c nh hng mot cach thch hp va tiep can hn vi lien ket ester va v vay chung b phan huy nhanh hn nhng ester co kha nang quay t do hn va cau truc t cng hn. Nhieu hp chat loai nay co cac goc lien ket cang thang va co the xem chung giong nh nhng lo xo ma mc o cang thang cua chung co the lam giam mot phan khi chung tham gia trang thai chuyen tiep. T cac dan lieu ve toc o nay co the xac nh c rang nong o Hieu lc cua cac nhom carboxyl xung quanh cac nhom ester co the cao en 105 - 108 M. o la nong o khong the co ve mat vat ly, song no cho phep minh hoa tnh u viet cua phan ng noi phan t oi vi phan ng gia cac phan t va cho thay viec mang cac chat phan ng lai gan nhau tai trung tam hoat ong cho phep tang toc o phan ng nhanh en mc nao. Bang 7. Cau truc va toc o tng oi (Vr ) cua cac phan ng thuy phan cac ester monophenyl cua cac ion acid dicarboxylic.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 40 -

* R = p-Br-C6 H4 - ; # toc o phu thuoc vao ban cat cua R 4. Gay mat on nh (Destabilization). Mot gia thuyet tng oi cu ve xuc tac enzyme cho thay rang s lien ket cua trung tam hoat ong lam cho cac lien ket b cang, bien dang hoac mat on nh nen de b t khi hnh thanh mot san pham hoac phc he trung gian ma trong o cac lien ket b tac ong tr nen kem ben vng hn so vi trong chat phan ng ban au. V du minh hoa cho gia thuyet nay la phan ng do base xuc tac khi thuy phan ethylene-phosphate xay ra nhanh gap 107 lan so vi khi thuy phan dimethyl-phosphate. H H CH3 O CH3 O H C C H O O

O O Ethylene-phosphate

O O Dimethyl-phosphate

Gia thuyet ve tnh cang nay co ve la mot cach giai thch hap dan oi vi xuc tac enzyme. Mot trong nhng v du thiet thc la tnh chat cua esterase cua gan. e thuy phan mot loat cac ester cua acid m-hydroxy-benzoic, Km hau nh khong phu thuoc vao chieu dai cua chuoi R, trong khi o Vmax tang len mot so bac khi chieudai cua chuoi tang. V lien ket chu s thuy phan la nh nhau trong moi trng hp, nen co the suy ra rang tang nang lng lien ket cuacac ester mach dai lam giam nang lng hoat hoa cua phan ng, tc la nang lng cua lien ket chat hn cua goc

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 41 -

hydrocarbon c bu ap bi sc cang c cam ng trong phan cha nhomacyl cua phan t. S gay mat on nh c chat cung co the xay ra khi cac c chat tch ien dang hoa tan, tc khi ket hp vi trung tam hoat ong, nhng c chat nay co the c chuyen t moi trng nc vao moi trng tng oi k nc trong trung tam hoat ong, nh o cho phep co s gia toc ln. Mo hnh phan ng dung e minh hoa hieu ng nay la phan ng decarboxyl-hoa mot dan xuat cua pyruvate va mot ong dang cua thiaminepyrophosphate xay ra nh sau:

Dan xuat nay (I) tng oi on nh trong nc va phan ng decarboxyl-hoa xay ra cham, nhng trong ethanol no b decarboxyl-hoa 104105 lan nhanh hn e tao ra CO2 va dan xuat III. Ngi ta cho ra cho rang toc o decarboxyl-hoa tang len la do trang thai trung gian gia nh (II) ien tch khu vc b giam so vi trong dan xuat I. ay la mot dan chng cho thay rang kieu nang toc o nay co the xay ra oi vi pyruvate decarboxylase ma cofactor la thyamine pyrophosphate v cofactor nay co the nam tai khu vc co tnh k nc tng oi cua enzyme. 5. Xuc tac acid-base phoi hp. Co nhieu phan ng xuc tac bang acid hoac bang base trong hoa hu c, v du s hnh thanh semiacetal c xuc tac bi acid hoac base. Base OH- thuc ay s hnh thanh semiacetal nh sau: OH CH3 C=O + CH3OH C CH3

H Acetaldehyde

Methanol

H OCH3 Semiacetal

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 42 -

Xuc tac bang acid dan en hnh thanh muoi oxonium e sau o tiep tuc phan ng vi alcohol:

Nhng dan chng co c hien nay cho phep gia thiet rang nhieu nhom trong trung tam hoat ong cua enzyme co the thc hien xuc tac theo kieu acid hay base oi vi cac nhom khac nhau trong c chat va bang cach o tham gia ay nhanh toc o phan ng. Xuc tac phoi hp acid-base ac biet co hieu qua va mot v du minh hoa la phan ng chuyen quay cua tetramethyl-glucose xay ra nh sau:

Acid va base eu xuc tac cho s chuyen quay. Chang han, khi mot trong hai dang anomer hoa tan trong benzen va them vao hon hp cua phenol va pyridine th qua trnh chuyen quay xay ra rat nhanh. ong hoc cua s chuyen quay cho thay rang toc o phu thuoc vao nong o cua phenol va pyridine cung nh cua tetramethylglucose. ieu o cho phep gia thiet rang phenol va pyridine hoat ong nh cac chat xuc tac acid va base mot cach ong thi. Hn the na, neu cac nhom chc nang cua phenol va pyridine cung nam trong mot phan t duy nhat, v du nh trong -pyridon (hydroxylpyridine) th se co c mot chat xuc tac co hieu qua cao hn nhieu, mac du tham ch cac nhom xuc tac trong -pyridon la nhng acid va base yeu hn nhieu so vi phenol va pyridine. Ngi ta cho rang -pyridon the hien tac dung xuc tac nh sau:

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 43 -

Nh vay, hydro tren nit cua pyridine (mot acid) cho proton, con oxy cua nhom carbonyl (mot base) nhan proton khi vong pyranose cua -anomer b m. Nit cua pyridine nhan proton t san pham trung gian co cau truc m, trong khi o nhom hydroxyl phenol cho proton khi vong ong lai e tao ra dang -anomer. Kieu xuc tac acid-base phoi hp nay xay ra trong cac c che xuc tac cua mot so enzyme, trong o co ribonuclease. Rat t co kha nang la kieu xuc tac nay lam tang toc o phan ng cao hn bac 10 hoac bac 100, nhng cung vi cac c che khac no ong gop vao viec nang cao toc o cua cac phan ng enzyme. Nhieu goc R cua aminoacid hoat ong nh kieu xuc tac phoi hp acidbase trong enzyme, bao gom cac goc acid glutamic, acid aspartic, histidine, lysine, tyrosine va cysteine. dang proton-hoa chung la nhng chat xuc tac acid, con dang khong proton-hoa chung la nhng chat xuc tac base. Ro rang la hieu qua cua cac goc R trong viec xuc tac se phu thuoc vao pK cua moi nhom chc nang va pH ma tai o xay ra phan ng enzyme. Bang 8 gii thieu mot so enzyme ma trong qua trnh xuc tac tao ra cac phc he enzyme-c chat trung gian lien ket ong hoa tr Thong thng nhng chat trung gian nay hoan toan co the c xac nh. V du, phc he acetyl-chymotrypsin hnh thanh trong qua trnh thuy phan pnitrophenylacetate bang chymotrypsin la mot chat ben vi pH acid va a c thu nhan dang che pham. O O - C CH3 O O - C - CH3 + O2N

O2N

HO C

OH

p-Nitrophenylacetate chymotrypsin

Acetyl-enzyme

p-Nitrophenol

Cac nhom thiol cua cystein trong cac enzyme thuy phan protein papain va ficin cung tham gia tao cac chat trung gian ong hoa tr vi c chat. Thioester gan vi enzyme c hnh thanh cung vi nhom carboxyl cua lien ket peptide nh sau:

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme
O Enz-SH + R-C-NH-R' O Enz-S-C-R + H2O O Enz-S-C-R + H2N-R' O Enz-SH + R-C-OH

- 44 -

Bang 8. Mot so enzyme ma trong c che xuc tac co s hnh thanh san pham trung gian enzyme-c chat lien ket ong hoa tr.
Loai enzyme Chymotrypsin,trypsin,subtil isin, elastase, thrombin, plasmin, acetylcholin esterase Phosphoglucomutase, phosphatase kiem Nhom phan ng HO-CH2-CH Serine Kieu sp. trung gian ong hoa tr O R-C-O-CH2OH Acyl ester O - O-P-O-CH2 -CH OPhosphoryl ester O R-C-S-CH2 -CH Acyl thioester -CH2 -CH O -O-P-N ON

HO-CH2-CH Serine HS-CH2-CH Cystein -CH2 - CH HN N

Papain, ficin, glyceraldehyde phosphate dehydrogenase Suxinic thiokinase, glucoso-6-phosphatase

Histidine Aldolase, transaldolase, pyridoxalphosphate enzyme NH2 -[CH2]4 -CH Lysine

Phosphoimidasole R R-C=N-[CH2]4 -CH Base Schiff

Mot dang lien ket ong hoa tr khac gia enzyme va c chat la trng hp hnh thanh base schiff (aldimine) gia cac hp chat carbonyl vi nhom -amine cua lysine trong mot so enzyme. V du, khi dihydroxyacetone phos- phate c u vi aldolase ma khong co mat glyceraldehyde-3-phosphate th phan ng sau ay se xay ra: CH2OH Enz-(CH2)4 -NH2 + O =C CH2O P CH2OH Enz-(CH2)4 -NH =C + H2O CH2O P

Tnh u viet chu yeu cua s hnh thanh cac chat trung gian ong hoa tr enzyme-c chat la cho chung lam tang xac suat e mot phan ng xac

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 45 -

nh co the c thc hien. Mot chat trung gian lien ket ong hoa tr vi enzyme buoc c chat b giam ham chat che ben trong trung tam hoat ong va co the c at vao t the thuan li hn cho phan ng ke tiep vi cac nhom thch hp tai trung tam e hoan thanh phan ng. Mc o thuc ay toc o bang cach tao ra cac chat trung gian ong hoa tr co the kha ln, v du oi vi aldolase, song trong cac trng hp khac no co the khong ln hn mc 102 -103 .

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme XIII. ISOENZYME

- 46 -

Nhieu enzyme trong cung mot c the, tham ch trong cung mot te bao, ton tai nhieu dang phan t khac nhau. Nhng dang phan t o cua cung mot enzyme c goi la isoenzyme. Chung thng c tach khoi nhau mot cach de dang bang phng phap ien di. V du lactate dehydrogenase trong cac mo cua chuot bach co 5 dang isoenzyme ma ngay nay a tach c dang tinh khiet. Chung eu xuc tac cho mot phan ng nh nhau, song gia tr Km cua chung rat khac nhau. Ca 5 dang nay eu co trong lng phan t vao khoang 134.000 va cha 4 chuoi polypeptide (trong lng phan t cua mot chuoi la 33.500). 5 isoenzyme la 5 kieu phoi hp khac nhau cua hai loai chuoi polypeptide chuoi M va chuoi H. Mot isoenzyme chiem u the trong c c cau tao t 4 chuoi M giong het nhau (ky hieu la M4). Isoenzyme th hai chiem u the trong tim, ky hieu la H4, c hnh thanh t 4 chuoi H. Ba isoemzyme con lai, ky hieu la M3H, M2H2 va MH3. Ngi ta a tach rieng c cac chuoi M va H. trang thai nay chung khong con kha nang xuc tac. Hai loai chuoi co thanh phan va trat t aminoacid khac nhau. Khi tron trong ong nghiem hai loai chuoi vi t le tng ng cac kieu isoenzyme khac nhau se t ong xuat hien. S tong hp cac chuoi M va H c ma hoa bi hai gen khac nhau, Nh vay, t le tng oi cua chung trong moi loai isoenzyme c kiem tra mc o gen va co the bien oi trong qua trnh phat trien cua phoi. Viec nghien cu isoenzyme co y ngha rat quan trong oi vi viec tm hieu c s phan t cua s phan hoa te bao va phat sinh c quan. Ngi ta cho rang khong nhng enzyme ma nhieu loai protein cung ton tai trong te bao cac dang khac nhau, phan biet nhau bi t le gia cac chui polypeptide von c ma hoa bi cac gen khac nhau.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme XIV. CAC NHOM ENZYME

- 47 -

1.Enzyme oxy hoa - kh. Trong cac phan ng oxy-hoa - kh sinh hoc ien t t c chat c van chuyen en oxy khong kh theo tng bc tren c s giam dan the kh tieu chuan. Moi enzyme oxy hoa - kh hoat ong nh s phoi hp cua mot nhom chc nang goi la cofactor hoac coenzyme. Mac du co hang tram loai enzyme oxy hoa - kh khac nhau tham gia trong cac qua trnh oxy-hoa sinh hoc, song ch co mot so rat t cac cofactor hoac coenzyme lam nhiem vu nhan hoac nhng ien t gia c chat va san pham. V du, ngi ta a phat hien c tren 250 enzyme eu s dung cac coenzyme nicotinamide nucleotide (NAD+ hoac NADP+) lam chat nhan ien t. Chung xuc tac cho cac phan ng co dang di ay: AH2 + E.NAD+ va AH2 + E.NADP+ A + E.NAD.H + H+ A + E.NADP.H + H+

The kh cua c chat (AH2) c chuyen cho NAD.H (hoac NADP.H) e sau o se tham gia trong cac qua trnh oxy hoa - kh khac. Mot so dehydrogenase khac s dung cac flavine nucleotide (F) lam coenzyme va xuc tac cac phan ng co dang tong quat sau ay: AH2 + E.F E.FH2 + A Enzyme dang kh (E.FH2) co the van chuyen ien t va proton cua no cho cac chat khac (v du trong qua trnh phosphoryl hoa oxy-hoa) hoac cho O2 e tao ra H2O2 : E.FH2 + O2 E.F + H2O2 . Xuc tac cho cac phan ng loai nay la cac enzyme thuoc nhom oxydase. Cofactor cua cac enzyme loai nay co the la cac ion kim loai nh Cu2+, Fe2+ hoac cac hp chat hu c tng oi phc tap ma chung ta se xet en sau. Cac enzyme oxy hoa - kh cua chuoi van chuyen ien t trong ty the va trong luc lap cung co cofactor la kim loai (Fe, Cu, Mo...), heme hoac cac hp chat hu c cha sat va lu huynh. Thong thng ch co mot nhom chc nang cua mot enzyme oxy hoa kh ket hp vi mot chuoi polypeptide e tao ra mot n v hoat ong. Tuy nhien, nhieu enzyme cha mot tap hp cac nhom chc nang, bao gom flavin nucleotide, cac nhom cha sat va lu huynh, cac nhom heme va ca kim loai e tao nen mot chuoi van chuyen ien t vi chieu dai va mc o phc tap khac nhau e ap ng cac nhu cau ac biet cua trao oi chat.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 48 -

a/ Dehydrogenase phu thuoc pyridine. Dehydrogenase phu thuoc pyridine la nhom enzyme oxy hoa - kh ma coenzyme la mot trong cac dan xuat cua pyridine. hai coenzyme pho bien nhat cua nhom dehydrogenase nay la nicotinamide adenine dinucleotide (NAD) va nicotinamide adenine dinucleotide phosphate (NADP). Cong thc cau tao cua chung c gii thieu trong hnh 12.

Hnh 12. Cong thc cau tao cua NAD+ va NADP+ Nhom dehydrogenase nay lam nhiem vu van chuyen thuan nghch tng oi ng lng kh (oi ien t hoac oi ien t cung vi oi proton) t c chat AH2 en dang oxy hoa cua coenzyme (NAD+ hay NADP+). Mot trong hai ng lng o dang hydro nam trong pyridine nucleotide kh (NAD.H hay NADP.H), con ng lng kia - dang ien t. Nguyen t hydro th hai sau khi tach khoi c chat c chuyen vao moi trng dang ion H+ t do (hnh 13).

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme
NAD+ (hay NADP+) NADH (hay NADP.H)

- 49 -

Hnh 13. Phan ng dehydrogenase phu thuoc pyridine. Nhng dehydrogenase lien quan vi NAD chu yeu tham gia qua trnh ho hap tc qua trnh van chuyen ien t t c chat en oxy. Trong khi o, cac enzyme lien quan vi NADP chu yeu tham gia van chuyen ien t t c chat tham gia phan ng d hoa en cac phan ng kh cua qua trnh sinh tong hp. V vay, phan ln NAD c phat hien trong ty the, con a so NADP th nam trong phan hoa tan cua te bao chat. a so dehydrogenase phu thuoc pyridine ch ac hieu vi NAD hay ch ac hieu vi NADP. Tuy nhien co mot so dehydrogenase (v du glutamate dehydrogenase) co the s dung ca hai coenzyme nay. Trong bang 9 gii thieu mot so dehydrogenase phu thuoc pyridine va ga tr Eo' cua oi c chat chu tac dung cua chung. Chieu hng cua phan ng va thanh phan can bang cua he thong oxy hoa - kh do nhom enzyme phu thuoc pyridine xuc tac co the d oan tren c s the kh tieu chuan cua oi NADH-NAD+ (hay NADP.H-NADP+) ma Eo' cua chung bang -0,32V. Neu qua trnh oxy hoa - kh c thc hien trong ieu kien tieu chuan th he thong co gia tr am cua the kh tieu chuan cao hn so vi NAD va NADP, se co xu hng nhng ien t cho dang oxy-hoa cua nhng coenzyme nay, con nhng he thong co gia tr dng cua the kh tieu chuan ln hn se co xu hng nhan ien t t NADH hay NADPH.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 50 -

Bang 9. The kh tieu chuan cua mot so he thong dehydro-genase phu thuoc pyridin. He thong Eo' cua oi c chat, (V) Phu thuoc NAD Isocitrate dehydrogenase - 0,38 D--oxybutyratedehydrogenase - 0,32 Glyceraldehyde-3-phosphate - 0,29 dehydrogenase Dihydrolipoil dehydrogenase - 0,24 Alcohol dehydrogenase - 0,20 Lactate dehydrogenase - 0,19 L-malate dehydrogenase - 0,17 Phu thuoc NADP Isocitrate dehydrogenase - 0,38 Glucoso-6-phosphate dehydrogenase - 0,32 Phu thuoc NAD hoac NADP L-glutamate dehydrogenase - 0,14 Nhieu enzyme thuoc nhom dehydrogenase phu thuoc pyridine thng ton tai mot so dang isoenzyme khac nhau, trong o cac cau truc di n v phoi hp theo cac ty le khac nhau. V du ien hnh la trng hp cua lactate dehydrogenase. Enzyme nay cha hai loai phan di n v ky hieu la H va M. Trong te bao a phat hien 5 loai isoenzyme vi 5 kieu phoi hp khac nhau gia hai loai phan di n v nay. Do co cau truc di n v khac nhau, nen moi dang isoenzyme cung phan biet nhau bi cac gia tr Km va Vmax ac trng trong quan he vi moi loai c chat va ong cac vai tro khac nhau trong qua trnh trao oi chat. NAD khong ch ong vai tro nh mot coenzyme trong cac phan ng oxy hoa - kh ma con co the tham gia trao oi chat te bao vi cac chc nang khac. V du, no la mot yeu to khong the thieu c trong phan ng do ADN ligase cua E. coli xuc tac. Trong phan ng nay NAD b phan huy thanh AMP va nicotinamide mononucleotide (NMN) e cung cap nang lng cho s hnh thanh lien ket phosphodiester gia hai oan polydeoxyribonucleotide ma ADN ligase co nhiem vu phai noi lai. b/ Dehydrogenase phu thuoc flavin. Dehydrogenase phu thuoc flavin (hay con goi la flavoprotein) la nhng enzyme ma coenzyme la riboflavin-5'-phosphate (flavin mononucleptide, FMN) hoac flavin adenine dinucleotide (FAD) ma cau truc cua chung c gii thieu trong hnh 14. S ket hp cua coenzyme vi apoenzyme trong cac

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 51 -

enzyme khac nhau c thc hien bang cac kieu lien ket khac nhau - lien ket ong hoa tr hoac lien ket khong ong hoa tr. Tuy nhien, ngay trong cac trng hp lien ket khong ong hoa tr th s ket hp gia coenzyme va apoenzyme van luon luon chat che hn so vi cac enzyme phu thuoc pyridine. Ngoai ra, cac enzyme flavine con co the cha mot hoac vai ion kim loai, phc he sat-lu huynh hoac heme e gay nen nhng bien oi ang ke trong hoat tnh xuc tac cua chung. Thuoc nhom flavoprotein quan trong nhat la nhng enzyme sau ay:

Hnh 14. Cong thc cau tao cua FMN va cua FAD . - NAD.H dehydrogenase: xuc tac s van chuyen ien t t NAD.H en mot chat nhan nao o cha c xac nh, co the la mot protein cha sat nao o trong chuoi ho hap. - Succinate dehydrogenase: xuc tac phan ng oxy-hoa acid suxinic thanh acid fumaric. - Dihydrolipoyl dehydrogenase cua he thong pyruvate dehydro-genase va -cetoglutarate dehydrogenase. beo. - Cac flavoprotein xuc tac giai oan au cua qua trnh -oxy-hoa acid

Bo phan hoat ong cua phan t FAD va FMN tham gia trong phan ng la vong isoaloxasine cua riboflavin. Phan ng c thc hien bang cach van chuyen trc tiep oi nguyen t hydro t c chat en FAD hoac FMN e tao ra dang kh cua coenzyme, tc FAD.H2 hoac FMN.H2 (hnh 15)

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 52 -

FAD

FAD.H2

Hnh 15. Phan ng dehydrogenase phu thuoc flavin Trong te bao chat nhan ien t t dehydrogenase phu thuoc flavin thng la mot so enzyme thuoc nhom cytochrome. c/ Cytochrome. Cytochrome la mot nhom protein cha sat co cau tao tng t nh hemoglobin, tham gia trong qua trnh van chuyen ien t trong ho hap cung nh trong quang hp. Trong qua trnh ho hap cytochrome am nhan viec van chuyen ien t t cac enzyme flavin en oxy khong kh; trong quang hp cytochrome tham gia trong van chuyen ien t pha sang. Cytochrome giong hemoglobin va myoglobin cho nhom them cua chung la cac hp chat porphyrin cha sat. Trong qua trnh xuc tac xay ra s bien hoa thuan nghch gia Fe3+ va Fe2+. Cytochrome ng cuoi cung trong chuoi ho hap co kha nang kh trc tiep oxy phan t thanh O2-, v vay no thng c goi la cytochrome oxydase. Cytochrome c tm thay trong moi c the hieu kh. Hn the na, ham lng cua chung trong cac c quan khac nhau co quan he chat che vi hoat ong ho hap cua cac c quan o. V du, c tim rat giau cytochrome, nhng trong gan, than, nao va ac biet la da, phoi ham lng cytochrome rat thap. Cac cytochrome khac nhau c phan biet tren c s quang pho hap thu va c ky hieu bang cac ch cai a, b, c hoac bang cach ghi chu kem theo gia tr cua bc song hap thu cc ai. (bang 10). Trong ty the cua thc vat va ong vat bac cao a tm thay hang loat cytochrome khac nhau: cytochrome a, cytochrome a3, cytochrome b, cytochrome b2 , cytochrome c va cytochrome o. Hang loat cytochrome khac cung c tm thay trong thanh phan cua chuoi van chuyen ien t trong thylacoid cua luc lap.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme
Bang 10. Tnh chat cua mot so cytochrome cua ong vat co vu. Cytochrome nh hap thu cua cac dang kh , nm , nm , nm a3 600 ... 445 a 605 517 414 c 550 521 416 c1 554 523 418 b 563 530 430 b1 565 535, 528 430 b5 557 527 423

- 53 -

Eo(mV) +200 +340 +260 +225 +30 -30 (+245) +0,03

Cytochrome thc hien viec van chuyen ien t vi s tham gia trc tiep cua nguyen t sat trong thanh phan cua nhom heme nam tai trung tam hoat ong cua moi cytochrome. Nhom prostetic cua hau het cac cytochrome, tr cytochrome a va cytochrome a3, eu la phc he gia protoporphyrin IX vi sat nh trong hemoglobin. Trong ty the, cac ien t bat nguon t dang kh cua cac enzyme dehydrogenase phu thuoc NAD hoac flavoprotein c nguyen t sat trong thanh phan cua heme cua mot cytochrome tiep nhan e sau o lai c chuyen cho mot nguyen t sat cua mot cytochrome khac. Trat t cua chuoi van chuyen nay se c e cap en sau. a so cytochrome gan kha chat vi mang. Nhieu cytochrome phoi hp chat che vi nhau va vi cac yeu to van chuyen ien t khac, tao nen cac cau truc gom nhieu phan di n v e khong nhng thc hien chc nang van chuyen ien t, ma con tham gia vao hoat ong bm proton de tao ra gradient proton von can cho viec tong hp ATP trong qua trnh phosphoryl hoa oxy hoa. 2. Transferase. Nhom enzyme transferase co ma so bang 2, c chia thanh 8 phan nhom sau ay: 2.1. enzyme xuc tac van chuyen cac nhom mot carbon (methyl, carbocyl, formyl); 2.2. enzyme van chuyen cac goc aldehyde va cetone; 2.3. Cac acyltransferase van chuyen cac goc acid, v du goc acetyl hoac suxinyl HOOC-CH2-CH2-CO-; 2.4. Cac enzyme thuoc nhom glycosyltransferase; 2.5. Cac transferase van chuyen cac chc ru; 2.6. Cac transferase van chuyen cac nhom cha nit (amin, amide, oximine) 2.7. Cac transferase van chuyen cac nhom cha phosphore; 2.8. Cac transferase van chuyen cac nhom cha lu huynh.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 54 -

Moi phan nhom nay tuy thuoc vao ban chat hoa hoc chua nhom c van chuyen lai c chia thanh cac phan nhom nho hn. Can lu y rang cac phan nhom transferase khac nhau co cac coenzyme khac nhau. V du cac aminotransferase co coenzyme la pyridoxalphosphate. Khi van chuyen cac nhom mot carbon en glycine enzyme la mot pteroptotein, con cac enzyme van chuyen cac goc ng th coenzyme la cac nucleotide. Nh vay, mac du nhom transferase tap hp cac enzyme van chuyen mot nhom hoa hoc nao o t phan t nay en phan t khac nhng phu thuoc vao ban chat hoa hoc cua nhom c van chuyen ma ban chat hoa hoc cua coenzyme la hoan toan khac nhau. Trong bang 11 gii thieu mot so v du ve nhom enzyme nay. Bang 11. Cac enzyme pho bien thuoc nhom transferase
Ma so 2.2.1.1 Ten he thong Ten thng dung
Sedoheptuloso-7phosphate:glyceraldehyde Transcetolase -3-phosphate glycolaldehyde transferase

2.4.1.1

1,4--DGlucan:ortophosphate-D-glucosyltransferase UDPlgucose:D-fructose 2--Dglucosyltransferase L-Aspartate:2cetyoglutarate aminotransferase L-Tyrosine:2cetoglutarate aminotransferase ATP : D-Hexoso-6phosphotransferase

Phosphorylase

2.4.1.1 3 2.6.1.1

Sacchrososyntase

phan ng Sedoheptuloso-7phosphate + D-Glyceraldehyde-3-phosphate = Driboso-5-phosphate+ DXyluloso-5-phosphate. (1,4---D-glucosyl)n + ortophosphate = (1,4---Dglucosyl)n-1 + (1,4---Dglucoso-1-phosphate UDP-glucose + D-Fructose = UDP + Saccharose L-Aspartate+ 2cetoglutarate = Oxaloglutarate + LGlutamate L-Tyrosine+ 2cetoglutarate = 4hydroxyphenylpyruvate = L-glutamate ATP + D-Hexose = ADP + D-Hexoso-6-phosphate

Aspartate aminotransferase

2.6.1.5

Tyrosinotransferase

2.7.1.1

Hexokinase

Trancetolase von co hoat tnh rat cao trong nam men va cay xanh, xuc tac cac phan ng van chuyen goc aldehyde glycolic t sedoheptuloso-7phosphate en D-glyceraldehyde-3-phosphate e tao ra D-riboso-5phosphate va D- xyluloso-5-phosphate.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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Nh vay, tranxetolase ong vai tro rat quan trong trong trao oi chat, xuc tac s chuyen hoa tng ho gia cac monosacharide. Phosphorylase xuc tac s van chuyen cac nhom glycosyl gia cac polyglycoside khac nhau (v du tinh bot hoac glycogen) va phosphate vo c theo phng trnh ghi trong bang 11. Phosphorylase trong cac mo ong vat la cac enzyme phan giai, trong khi o cac enzyme tng ng nguon goc thc vat co kha nang tong hp tinh bot. Enzyme tiep theo trong bang 11 la saccharosyntase. ai dien cua nhom enzyme nay hoat ong vi s tham gia cua cac nucleoside diphosphate. Aspartate aminotransferase la mot ai dien cua phan nhom aminotransferase, xuc tac phan ng van chuyen nhom amin t acid asparaginic en acid -cetoglutaric. Tyrosinoaminotransferase co tnh ac hieu kha rong vi hang loat aminoacid. V du no co the xuc tac phan ng chuyen amin hoa gia acid cetoglutaric va phenylalanine, gia 3,4-dioxyphenylalanine va trytophane. Hexokinase xuc tac phan ng phosphoryl hoa glucose, mannose va fructose tai nguyen t carbon th 6. 3. Hydrolase. Tnh chat chung cua nhom enzyme nay la xuc tac cac phan ng thuy phan tc phan giai cac hp chat phc tap vi s tham gia cua nc thanh nhng hp chat n gian hn. Hydrolase c chia thanh 11 phan nhom: 3.1: Enzyme phan giai cac lien ket ester; 3.2: Enzyme phan giai cac lien ket glycoside; 3.3: Enzyme phan giai cac lien ket eter; 3.4: Enzyme phan giai cac lien ket peptide; 3.5: Enzyme phan giai cac lien ket C-N khong phai peptide, v du cac amide; 3.6: Enzyme phan giai cac lien ket anhydride acid; 3.7: Enzyme phan giai cac lien ket C-C; 3.8: Enzyme phan giai cac lien ket haloid ru; 3.9: Enzyme phan giai cac lien ket P-N; 3.10: Enzyme phan giai cac lien ket S-N; 3.11: Enzyme phan giai cac lien ket C-P. Chung ta hay xem xet mot so hydrolase quan trong nhat.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 56 -

- Triacylglyceroyl-lipase enzyme xuc tac cac phan ng thuy phan triacylglycerin, ma so 3.1.1.3. Ten thng goi la lipase. Nhom 3.1.1.3 tap hp cac enzyme lipase khac nhau. V du lipase cua hat thau dau khong tan trong nc, co pHopt 4,7-5,0. No khong nhng xuc tac phan ng phan giai triacylglycerin ma xuc tac ca phan ng ngc tong hp triacylglycerine t glycerine va acid beo. Lipase tuyen tuy tan trong nc, co pHopt trong vung kiem yeu. Cac loai lipase khac, v du lipase vi sinh vat va lipase trong phoi lua mach, khac vi lipase hat thau dau cho tan trong nc co pHopt trong vung kiem yeu vi pH bang 8. ai dien cho phan nhom 3.2 la cac enzyme: --Amilase (3.2.1.1) cha trong nc bot, hat thoc nay mam, tuyen tuy, nam moc va vi khuan. No phan giai tinh bot thanh cac dextrin va mot lng nho ng maltose. - -Amylase (3.2.1.2) phan giai tinh bot chu yeu thanh ng maltose va mot t destrin phan t ln. - Exo-1,4-D-glucoamylase (3.2.1.3) vi ten thng goi la glucoamylase phan giai tinh bot thanh ng glucose va mot t dextrin. va -amylase khac nhau ve pHopt va kha nang chu nhiet: -amylase hoat ong trong ieu kien pH acid hn va chu nhiet tot hn so vi amylase. Che pham exo-1,4-glucoamylase c thu nhan t nam moc. V no phan giai tinh bot thanh glucose nen tr thanh mot enzyme c ac biet quan tam v nh no co the thu nhan che pham glucose ma khong can s dung phng phap thuy phan bang acid. Glucoamylase thng c thu nhan t nam Aspergillus niger, Aspergillus usamii, Rhisopusniveus. pHopt cua no nam trong khoang 4,5-4,7. Mac du glucoamylase co the thuy phan cac lien ket -D-(1-4), -D-(1-6) va -D-(1-3) glycoside nhng thuy phan cac lien ket -D-(1-4) xay ra nhanh hn nhieu. - -D-Fructofuranosidase (3.2.1.26) vi ten thng goi la invertase hay saccharase phan giai sacchasose thanh glucose va fructose, co nhieu trong nam nen von c dung e thu nhan che pham enzyme nay. Ngoai -DFructofuranosidase, sacchasose con c thuy phan bi -D-glucosidase (3.2.1.20): -D-Fructofuranosidase Thuy phan ca rafinose va metyl -Dfructofuranoside nhng vi toc o cham hn.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 57 -

Trong so cac enzyme thuy phan polycacharise con co cellulase (3.2.1.4) thuy phan cellulose thanh disaccharide celobiose. pHopt cua enzyme nay nam trong khoang 5,0-5,4. - Thuoc nhom hydrolase con can phai ke en cac enzyme phan giai protein, tc peptid-hydrolase (3.4). phan ng c ban do phan nhom enzyme nay xuc tac la phan giai lien ket peptide. Phan nhom enzyme nay rat c quan tam do y ngha thc te cua chung, ac biet la kha nang s dung chung trong nen kinh te quoc dan. 4. Liase. Nhom liase bao gom 7 phan nhom sau ay: 4.1 Bao gom cac enzyme xuc tac cac phan ng cat cac lien ket gia cac nguyen t carbon; 4.2 - Bao gom cac enzyme xuc tac cac phan ng cat cac lien ket gia cac nguyen t carbon va oxy; 4.3 - Bao gom cac enzyme xuc tac cac phan ng cat cac lien ket gia cac nguyen t carbon va nit; 4.4 - Bao gom cac enzyme xuc tac cac phan ng cat cac lien ket gia cac nguyen t carbon va lu huynh; 4.5 - Bao gom cac enzyme xuc tac cac phan ng cat cac lien ket gia cac nguyen t carbon va haloid; 4.6. Bao gom cac enzyme xuc tac cac phan ng cat cac lien ket gia cac nguyen t phospho va oxy; 4.99 Cac liase khac. Bang 12 gii thieu mot so enzyme quan trong thuoc 3 phan nhom au. Bang 12. Mot so enzyme thc nhom liase Ma so Ten enzyme Phan ng

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme
4.1.1.1 Pyruvate decarbocylase

- 58 -

2-Oxyacid = Aldehyde + CO2 L-Glutamate = 4Aminobutyrate + CO2 D-Ribuloso-1,5-diphosphate + CO2 +H2O = 2,3-phospho-Dglycerate D-Fructoso-1,6-diphosphate = D-Dioxyacetonephosphate + D-Glyceraldehyde-3-phosphate L-Malate = Fumarate +H2O L-Aspartate = Fumarate + NH3

4.1.1.15 Glutamate decarbocylase 4.1.1.39 Ribulosodiphosphate carbocylase

4.1.2.13 Fructosodiphosphate aldolase

4.2.1.2 4.3.1.1

Fumarate hydratase (Fumarase) Aspartate-ammoniac liase (Aspartase)

5. Isomerase. Nhom Isomerase bao gom 6 phan nhom: 5.1: Rasemase va epymerase; 5.2: Cis-trans-isomerase 5.3: Oxydoreductase not phan t; 5.4: Transferase not phan t; 5.5:Liase noi phan t; 5.99: Cac isomerase khac. Thuoc phan nhom 5.1 gom co cac enzyme xuc tac qua trnh ong phan hoa aminoacid (5.1.1), glucid va cac dan xuat cua chung (5.1.2). ai dien ien hnh cua phan nhom nay la alaninerasemase (5.1.1.1) xuc tac qua trnh chuyen hoa tng ho gia L- va D-alanine, lactaterasemase (5.1.2.1) xuc tac qua trnh chuywen hoa tng ho gia L- va D-acid lactic va UDP-glucose-4epimerase (5.1.3.2) xuc tac qua trnh chuywen hoa tng ho gia UDP-glucose va UDP-galactose. ai dien cho phan nhom 5.2 la maleinate isomerase (5.2.1.1) xuc tac s chuyen hoa tng ho gia acid fumaric va acid maleic. ai dien cho phan nhom 5.3 la triosophosphate isomerase (5.3.1.1) xuc tac s chuyen hoa tng ho gia D-glyceraldehyde-3phosphate va dioxyacetonphosphate.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 59 -

Phan nhom th t bao gom cac enzyme xuc tac cac phan ng transferase not phan t. Trong so cac ai dien cua nhom nay co the ke en methylaspartatemutase va S-methylmalonyl-CoA-mutase: 6. Ligase (synthetase). Nhom nay c chia lam 5 phan nhom: 6.1: Lagase tao cac lien ket C-O; 6.2: Ligase tao cac lien ket C-S; 6.3: Ligase tao cac lien ket C-N; 6.4: Ligase tao cac lien ket C-C; 6.5: Ligase tao cac lien ket phosphodiester. Thuoc phan nhom 6.1 la tat ca cac enzyme aminoacyltARNsyntase. Thuoc phan nhom 6.2 co 13 enzyme xuc tac s dung nap cac acyl khac nhau, v du acetyl hoac suxinyl vao coenzme , v du acetyl-CoAsynthetase (6.2.1.1) xuc tac phan ng: ATP + Acetate + CoA-SH Acetyl-CoA. AMP + Pyrophosphate +

Nh ta a biet, acetyl-CoA tham gia tong hp rat nhieu hp chat khac nhau, trong o co steroid va polyisoprenoid. Phan nhom 6.3 gom co 5 nhom enzyme, ai dien la asparagine synthetase (6.3.1.1) va glutamine synthetase (6.3.1.2), xuc tac cac phan ng tong hp asparagine va glutamine t cac aminoacid dicarboxylic tng ng vi s tham gia cua ATP. Phan nhom 6.4 gom 5 loai carboxylase cha biotin, xuc tac vi s tham gia cua ATP va CO2 cac phan ng carboxyl hoa cac acid hu c khac nhau. V du pyruvate carboxylase (6.4.1.1) xuc tac phan ng thuan nghch gia pyruvate va oxaloacetate. Phan nhom 6.5 co 2 enzyme xuc tac s khoi phuc cac lien ket phosphodiester b pha v trong acid nucleic, v du enzyme polydesoxyribonucleotide synthetase (ATP), ma so 6.5.1.1, xuc tac phan ng: ATP + (Desoxyribonucleotide)n + (Desoxyribonucleotide)m AMP + (Desoxyribonucleotide)n+m + Pyrophosphate.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme XV. TACH CHIET VA TINH CHE ENZYME.

- 60 -

Lam the nao e tach chiet va sau o tinh che enzyme t mot vat lieu sinh hoc? Giai oan au cua qua trnh tach chiet enzyme la thu nhan dch chiet t mot vat lieu sinh hoc nghien nat s bo nao o bot, mam thoc, hat nay mam, mot mo ong vat nao o, mot sinh khoi vi khuan hoac nam ... Trong dch chiet nay ng nhien co cha protein va enzyme. Trong viec nghien cu enzyme ngi ta thng hay s dung dch nghien nat ong the. e thu nhan loai dch nay khoi vat lieu sinh hoc c ra sach va nghien trong coi hoac trong mot thiet b chuyen dung co ten goi la may nghien. Trong khi nghien ngi ta them nc hoac dung dch em va knh ngien nat e giup pha v te bao. San pham thu c sau khi nghien c goi chung la dch ong the. Trong khoi dch nay cha cac manh te bao, nhan, luc lap cua la, ty the va cac bo phan khac cua te bao nh sac to, protein hoa tan v.v... Nghien nat te bao trong nc hoac trong dung dch em con co the c thc hien bang cach tac ong len chung bang sieu am trong mot thiet b ac biet goi la may nghien sieu am. Phng phap nay ngay nay c s dung rat rong rai, ac biet, e pha v te bao vi khuan. Tuy nhien khi s dung thiet b nay can phai chon che o lam viec sao cho enzyme khong b mat hoat tnh. Dch nghien te bao sau o co the c ly tam phan oan cac toc o tang dan khac nhau. V du, co the bat au ly tam 1500 g, tc gia toc cao hn gia toc cua trong lc 1500 lan. Sau khi tach c cac tieu phan lang oc xuong ay ong nghiem lai tiep tuc ly tam 20.000 g, 40.000 g v.v... 1500 g chloroplast re lang xuong ay, 20.000 g en phien lang ong cua ty the; 40.000 g hoac gia toc cao hn se lang ong nhng hat nho hn. e gi cho cac c quan t cua te bao (ty the, nhan v.v... co nguyen ven khi ngien te bao thc vat e ly tam thng can them vao khoi vat lieu can nghien 5 8% saccharose. Neu dung cach nay e tach khoi vat lieu mot phan oan cau truc di te bao nao o (luc lap, ty the v.v...) th enzyme cha trong chung co the con nam trong phan oan o dang lien ket. e tach chiet t chung va chuyen nhng enzyme o sang dang dung dch th can phai pha v cac cau truc di te bao o. e lam cong viec nay ngi ta thng dung cac chat tay ra detergent von la nhng chat co hoat tnh be mat rat cao. Neu c them vao dch nghien sinh khoi mot lng rat nho chung se lam pha v cac cau truc te bao va di te bao. Thng hay dung nhat la twin (ten thng mai cua hon hp sorbit va cac acid beo phan t ln) hay desoxycholate.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 61 -

Mot phng phap khac thng dung e tach chiet enzyme khoi cac cau truc di te bao la lien tuc lam ong bang va cho tan bang phan oan cau truc di te bao can nghien cu va sau o tien hanh ly tam phan oan. e thu nhan cac enzyme trong cac dch chiet hoac t cac phan oan di te bao co the dung nc, dung dch em, dung dch muoi (v du NaCl 10%) hon hp glycerine vi nc (v du 40% glycerine va 60% nc) hoac dung moi hu c (v du acetone). e tach chiet nhieu enzyme ngi ta con dung bot acetone. Nhng net c ban cua phng phap nay nh sau: khi nghien mot mau vat nao o, v du hat nay mam, cho them vao dch nghien vai lan acetone lanh e loai bo cac chat co ban chat lipid, mot so chat nha , chat co mau. Sau khi e kho thu c mot loai bot ong nhat. sau khi chiet xuat bang dung dch em hoac bang mot dung moi nao o se thu c dch chiet cha cac enzyme quan tam. ang tiec la phng phap nay khong s dung c cho tat ca enzyme v mot so enzyme b mat hoat tnh trong acetone Lam the nao tach c mot enzyme ma ta quan tam t dch chiet bang nc hoac bang dung dch muoi? Co nhieu phng phap khac nhau e thc hien cong viec nay. Co ien nhat la phng phap ket tua protein enzyme amylase t mam lua mach bang ethanol hoac acetone a c A. Payen va J. Perco thc hien t au the ky 20. Phng phap nay ngay nay c s dung kha rong rai. ac biet la e thu nhan enzyme t nam moc. V du, mot so cong ty cua Nhat san xuat che pham amylase t nam moc Aspergillus oryzae nh sau: nuoi trong Aspergillus oryzae trong moi trng dinh dng co thanh phan xac nh. Sau o sinh khoi nam c tach khoi moi trng c nghien va chiet xuat va t dch chiet nay tien hanh ket tua enzyme bang ethanol vi nong o xac nh. ng nhien, bang phng phap nay trong che pham enzyme thu nhan c khong ch co amylase ma co ca nhieu enzyme va protein khac. iem yeu c ban cua phng phap nay la khong phai moi enzyme eu chu ng c viec x ly bang ethanol va acetone. Mot so trong chung se b bien tnh va mat hoat tnh. e tranh thieu sot nay can phai thc hien viec ket tua enzyme t dung dch bang cac dung moi hu c nhiet o thap, gan vi nhiet o ong bang cua hon hp dung moi va nc. Ben canh phng phap ket tua enzyme bang cac dung moi hu c, e thu nhan cac che pham enzyme khac nhau ngi ta con s dung phng phap diem tch. Phng phap nay c s dung kha rong rai trong cong tac nghien cu khoa hoc e thu nhan cac che pham enzyme co hoat tnh cao. Phng phap diem tch hay dung nhat la s dung sulfate ammon (NH4)2SO4, them

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 62 -

vao dung dch nghien cu vi lieu lng tang dan. Dung dch cha enzyme trc tien c them vao sulfate ammon, v du en 20% mc bao hoa toan phan. Khi o mot phan protein va enzyme nao o se ket tua. Tach tua bang ly tam va nghien cu hoat tnh enzyme trong tua o. Dung dch tiep tuc c them sulfate ammon, v du en 40% mc bao hoa toan phan. Lai tach tua bang ly tam va nghien cu s ton tai cua hoat tnh enzyme. Phan dung dch cong lai sau khi ly tam lai tiep tuc c bo sung sulfate ammon en 60% mc bao hoa toan phan va lai tiep tuc ly tam e thu nhan enzyme nh mo ta tren. Nh vay, co the thu c hang loat phan oan protein e nghien cu s ton tai cua enzyme nay hoac khac trong chung. Mot phng phap tach chiet va tinh che enzyme rtat quan trong la phng phap hap phu chon loc t dung dch. Noi dung cua phng phap nay la dung dch cha enzyme c hap phu bi mot chat hap phu xac nh, v du hydroxide nhom. Phng phap hap phu chon loc c s dung rat rong rai trong cong nghiep enzyme e tach va tinh che amylase t nam va vi khuan. Trong cong viec nay chat hap phu thng c s dung la tinh bot von la mot chat hap phu ac hieu oi vi enzyme nay. Ngoai ra trong enzyme hoc con s dung hang loat chat hap phu khac thch hp vi tng loai enzyme. Mot phng phap rat tot dung e tach enzyme la phng phap loc gen. Thng hay c s dung nhat trong phng phap nay la sephadex. o la mot loai dan xuat cua dextran mot loai polysaccharide phan t ln cua mot so loai vi sinh vat Leuconostoc sinh trng va phat trien trong moi trng co saccharose. Phan t dextran c cau tao t cac chuoi, trong o cac goc glucose noi vi nhau bang lien ket glycoside 1 : 6. Trong lng phan t cua dextran at en mot trieu hoac cao hn. Sephadex c thu nhan t dextran bang phng phap x ly hoa hoc e tao ra cac lien ket ngang, lam cho san pham khong hoa tan trong nc nhng co the trng phong trong nc. S dung sephadex trong viec tach chiet protein va enzyme da tren c s nh sau. V du ta co mot cot sac ky cha sephadex. Cho vao cot mot hon hp gom hai chat co trong lng phan t khac nhau, v du muoi va protein. Khi loc qua cot gel sephadex cac chat phan t nho se di chuyen t t qua cac lo xop cua sephadex, con cac chat co trong lng phan t ln hn, ma trong trng hp nay la protein, se nhanh chong chay xuyen qua cac khe h gia cac hat sephadex. Nh vay, co the tach c hai chat vi trong lng phan t khac nhau. Neu sau o tien hanh ra cot bang chnh dung dch em dung trc o th se lay ra c chat con gi lai trong cot, trong trng hp nay la muoi, ong thi khoi phuc hoat ong cua cot sephadex. V c s cua phng phap

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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tach chiet nay la s khac nhau ve trong lng phan t nen phng phap loc gen con c thng gol la phng phap ray phan t. Ngay nay tren the gii ngi ta a san xuat nhieu loai sephadex vi kch thc hat va o phan nhanh khac nhau va do o co kha nang tach chiet khac nhau. Nhng san pham nay c ky hieu bang cac con so: G-10, G-25, G-50, G-75, G-100, G-200 v.v... V du dung cot sac ky cha sephadex G-75 co the tach c cac protein vi trong lng phan t 3-70.000; neu cha sephadex G-100 co the tach c cac protein vi trong lng phan t 4150.000, con neu cha sephadex G-200 th co the tach c cac protein vi trong lng phan t 5-800.000. e tach chiet va tinh che enzyme ngay nay s dung rat pho bien phng phap ien di tren cac loai gel khac nhau polyacrylamide, agarose, tinh bot v.v... bao hoa dung dch em. S dung gel ngoai kha nang loai bo anh hng cua tac dung oi lu con cho phep tach protein khong nhng theo ien tch ma theo ca trong lng va hnh dang phan t . Trong viec tach chiet va tinh che enzyme Tren the gii s dung ngay cang pho bien phng phap sac ky hap phu enzyme tren cot cha cac chat hap phu chon loc oi vi tng loai enzyme. Phng phap nay ac biet co gia tr trong viec tinh che enzyme. V du bang phng phap nay a tinh che enzyme L-asparginase vi hoat tnh cao gap 25-30 lan so vi cac phng phap tinh che khac. Phng phap sac ky hap phu enzyme va protein noi chung ang m ra nhieu con ng mi e hoan thien cong nghe hoa sinh va cong nghe hoa hoc va tao ra cac phng phap t ong hoa mi trong cac phan tch sinh hoa. Trong viec tach chiet va tinh che enzyme nhiet o thap co y ngha rat quan trong. nhiet o bnh thng cua phong th nghiem nhieu enzyme b bien tnh va mat mot phan hoac toan bo hoat tnh. V vay can lam viec vi enzyme bang cac thiet b at trong phong lanh. Ngay nay ai bo phan enzyme eu c thu nhan dang tinh the. oi vi nhng che pham nay can phai bao quan nhiet o thap, tot hn het la nhiet o ong bang. Cac dung dch enzyme tinh khiet hoac cac che pham enzyme dang tinh the co the c say kho bang cac phng phap khac nhau. Trong nhieu trng hp co the dung phng phap say kho chan khong vi s co mat cua cac chat hut nc nao o, ma tot nhat la P2O5. Nhng tot hn ca la la phng phap say lanh, say trang thai ong bang. Bang phng phap nay protein va enzyme gi rat tot tnh chat von co cua chung.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 64 -

Cho en nay khong co phng phap tach va tinh che chung cho cac enzyme. e tach va tinh che mot enzyme nao o can biet la chon va phoi hp mot cach co hieu qua nhat cac bien khac nhau.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme XVI. S DUNG ENZYME TRONG CONG NGHE SINH HOC.

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Cac che pham enzyme ngay cang co nhieu y ngha trong cong nghe sinh hoc. Chung c s dung khong nhng trong cac san xuat sinh hoa truyen thong nh lam banh my, lam bia, san xuat ru va cac loai nc hoa qua ma ca trong cac lnh vc cong nghe hoa sinh va vi sinh hoc. Cac che pham enzyme c s dung rat rong rai trong cac nghien cu sinh hoc vi t cach la cac hoa chat dung e nh lng cac hp chat khac nhau nh glucose, urea v.v... S ng dung ngay cang rong rai nhng kien thc nay a c ghi nhan trong lnh vc y hoc. Lan au tien nam 1884 cac che pham enzyme a c nha khoa hoc Nhat ban Takaminhe s dung quy mo cong nghiep. Ong a dung amylase cua nam moc Aspergiluc oryzae e ng hoa tinh bot. Cong trnh nay a co tac dung thuc ay s phat trien nhanh chong cua enzyme hoc ng dung. Ngay nay hang loat cac cong ty Nhat, Hoa ky, Anh, Phap va nhieu nc khac a san xuat cac che pham enzyme vi o tinh khiet cao e s dung trong cong nghiep, nong nghiep, trong thc tien phan tch va y hoc. Ta se xem xet mot vai v du ve ng dung cac che pham enzyme trong thc tien. Trong cong nghiep san xuat banh my them mot lng nho -amylase (0,002-0,003% trong lng bot) thu nhan t Aspergillus oryzae hoac Aspergillus awamori se lam tang ang ke hng thm va v ngon cua banh my, lam cho banh n eu va mau cua banh them am a. Lam tang hng v cua banh la ket qua cua qua trnh tang cng tao melanoidin di tac dung cua -amylase, lam cho cac hp chat carbonyl bay hi giup banh co mui thm tang len ang ke. Cac enzyme phan giai tinh bot c s dung rong rai trong viec ng hoa tinh bot khoai tay, hat ngu coc e san xuat ru. T thi xa xa ngi ta s dung bot mam ai mach e lam nguon amylase. tuy nhien mam ai mach co the c thay the bang nam moc Aspergillus oryzae. Viec thay the nay a thuc ay ang ke nghe san xuat ru. Mot so loai protease, nh papain, pepsin cung c s dung rong rai trong cong nghe san xuat bia e ngan nga hien tng bia b uc (ac bet khi bao quan bia trong ieu kien lanh) do hien tng co ten goi la lang tua protein-tannin gay ra. Cac enzyme phan giai protein cung c s dung trong viec lam mem tht. Tht thng gi o cng kha lau sau khi con vat b giet. e cho tht co c o mem can thiet, thng phai x ly trong ieu kien lanh 02o t 10

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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en 14 ngay. Trong ieu kien bao quan nh vay cung ch co 14-17% so tht c anh gia la tht loai I. Mot ieu kien quan trong nhat e lam xuat hien nhng chat lng can thiet cua tht la lam phan giai mot phan protein cua tht di tac dung cua cac enzyme proteinase. Ngat nay papain va cac enzyme proteolitic nguon goc vi khuan, nam, thc vat c dung pho bien trong viec x ly tht bao quan, chu yeu bang 3 phng phap sau ay: 1/ ngam trong dung dch enzyme; 2/ boi bot lam mem tht co cha enzyme cung vi m chnh, muoi an, tinh bot v.v..; 3/ Bm dung dch enzyme vao mo tht. Enzyme proteolitic c s dung co ket qua trong cong nghe thuoc da va lam mem da nguyen lieu. S dung enzyme lam tang ang ke chat lng cua len va lam nhanh qua trnh x ly. Qua trnh san xuat da, ac biet la qua trnh lam mem da nguyen lieu, hoan toan da tren viec s dung enzyme nguon goc ong vat, thc vat hoac vi sinh vat. ac biet s dung kha rong rai pancreatin che pham tho cua cac enzyme proteolitic cua tuyen tuy. Ngi ta cung s dung cac enzyme nguon goc thc vat (papain) va enzyme proteolitic thu t nam moc (Aspergillus oryzae, Aspergillus parasiticus) va vi khuan (Bacillus mesentericus). Trong cong nghiep san xuat nc trai cay thng s dung cac enzyme phan giai pectin. Chung lam tang ang ke mc thu hoach san pham (8-15%), giam o nht va lam cho nc trai cay trong hn. Trong cong nghe san xuat banh keo thng dung -fructofuranosidase. Van e la cho ng saccharose rat de b ket tinh, anh hng xau en chat lng san pham. Di tac dung cua -fructofuranosidase saccharose b phan giai, do o ngan can qua trnh ket tinh cua saccharose. Nhieu kinh nghiem cua cac nha san xuat cho thay cac che pham cellulase thu t nam moc trong o co cha hemicellulase co the s dung co hieu qua trong viec che bien thc an gia suc, lam tang ang ke o hap thu cua thc an, giup tang trong gia suc, giup cho ga e nhieu. Vi s ho tr cua cac che pham enzyme cellulase phoi hp tng t cac nha san xuat a thanh cong trong viec tang san pham tinh bot t cac nguyen lieu giau tinh bot, tang san pham agar-agar t cac nguyen lieu tao bien, tang lng nc trai cay t cam va cac loai trai cay khac. Viec nghien cu ng dung enzyme trong y hoc cung ngay cang thu c nhieu ket qua. Trong cac phong th nghiem y hoc ngi ta s dung cac loai dehydrogenase khac nhau e lam cac thuoc th ac biet, v du dung lactate dehydrogenase e xac nh acid pyruvic va acid lactic, s dung

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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alcohol dehydroganase e xac nh ethanol v.v... Cac bo che pham enzyme co cha alcoholdehydrogenase c s dung mot so nc e xac nh lng con trong mau cua lai xe. a nhieu nam troi qua ke t khi glucosooxydase c s dung rong rai trong y hoc e xac nh glucose trong nc tieu va trong mau. Mot lnh vc khac cua viec ng dung glucosooxydase la s dung no e loai bo glucose t cac san pham thc an phai bao quan trong ieu kien kho. V du, s ton tai glucose trong trng se dan en tnh trang bot trng che tao t loai trng nay co mui va v kho chu. o la do glucose khi say kho va bao quan nhiet o cao de tac dung vi cac nhom amine t do cua aminoacid va protein. bot sa se b en va tao ra hang loat cac chat co mui v kho chu. V vay trc khi say kho nguyen lieu trng can c loai bo glucose bang cach x ly glucosooxydase e san pham bot trng c ben vng trong qua trnh bao quan. O My ngi ta s dung glucosooxydase e loai bo oxy khoi o hop va nhieu loai nc giai khat khac nhau, v du bia.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme XVII. ENZYME CO NH.

1.Y ngha cua enzyme co nh.

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Trong nhng nam cuoi cua the ky 20 bat au pho bien ky thuat co nh enzyme. Enzyme hoa tan sau khi s dung thng lan vao cung vi san pham khong tach ra c. Neu tach ra th enzyme b mat hoat tnh, v vay vi mot lng enzyme nhat nh ch co the s dung c mot lan. S dung enzyme co nh co mot so u iem sau: - Mot lng enzyme co the s dung lap i lap lai c nhieu lan trong mot thi gian dai; - Enzyme khong lan vao trong san pham, do o tranh c anh hng khong tot en chat lng san pham; - Dung enzyme co nh co the dng nhanh chong phan ng khi can thiet bang cach tach no ra khoi c chat. 2. Cac phng phap ieu che enzyme co nh. Theo ky thuat che tao enzyme co nh, enzyme c gan vao mot vat mang nao o. Nhng vat mang nh vay co the la cellulose va cac dan xuat cua no, cac hat knh xop, gel polyacrylamide, sephadex, collodium, tinh bot, cac loai allumosilicate va oxide kim loai...Ve nguyen tac, co 3 phng phap ieu che cac enzyme co nh: - Gan bang lien ket ong hoa tr phan t enzyme vao chat mang khong hoa tan hoac gan cac enzyme vi nhau e tao nen ai phan t khong hoa tan (hnh 16);

Hnh 16. S o ieu che enzyme co nh bang cach nh mach ngang nh cac tac nhan lng chc hoac a chc.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

- 69 -

- nh enzyme len be mat chat mang hoac vao trong long khuon gel co kch thc lo kha nho u e gi enzyme, con e cac chat khac qua lai t do (Hnh 17);

Hnh 17. Hap hu enzyme tren be mat chat mang (a) va trong long chat mang (b)

- Hap phu enzyme len tren cac chat mang khong hoa tan co mang hoac khong mang ien tch (Hnh 18).

Hnh 18. S o hap phu enzyme tren chat mang khong co va co ien tch.

a/ Phng phap gan enzyme bang lien ket ong hoa tr. a so enzyme co nh thu c bang phng phap nay. Vi phng phap nay co the thu enzyme co nh bang hai cach. Ket hp phan t protein enzyme vao chat mang khong hoa tan. Cac chat mang e gan enzyme phai thoa man nhng yeu cau sau: Co o hoa tan thap va ben vng oi vi cac tac ong c hoc va hoa hoc; Khong gay tac ong km ham en hoat tnh enzyme; Khong hap phu phi chon loc oi vi cac protein khac; Chat mang tot hn ca la co tnh hao nc, v chat mang k nc co the gay tac dung c che enzyme a lien ket;

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme
-

- 70 -

Viec gan enzyme se co hieu qua hn khi ien tch cua enzyme va cua chat mang co dau ngc nhau.

Cac chat mang loai nay thng la polypeptide, cac dan xuat cua cellulose va dextran (DEAE-cellulose, CM-cellulose, DEAE- sephadex, CM, sephadex), agarose va cac polimer tong hp khac nh polyacrilamide, polystyrol, polyamide va cac chat vo c nh silicagen, bentonit, hydroxide nhom v.v... Chat mang pho bien hn ca la dextran co lien ket ngang (Sephadex) va agarose hat (sepharose). Cac hai loai chat nay eu co cau truc lo xop khien cho cac phan t ln co the xam nhap vao gel mot cach de dang. Poliacrylamide cung la chat mang rat tien li vi co o ben vng c hoc va hoa hoc cao. Chat mang vo c thng rat ben vi nhiet, c hoc, vi dung moi hu c va vi cac vi sinh vat, nhat la khong b bien oi cau hnh cua khuon khi thay oi tnh chat cua moi trng chung quanh. Nhieu dan lieu cho thay rang enzyme nh vi khuon vo c khi bao quan thng ben vng hn enzyme gan vi khuon polymer hu c. Tham gia tao thanh cac lien ket ong hoa tr co the co cac nhom chc cua phan t protein enzyme nh nhom - va -carboxyl COOH cua acid asparaginic va acid glutamic, nhom -NH2 cua lysine, nhom -SH cua cysteine, vong phenol cua tyrosine, nhan imidasol cua histidine, cac nhom OH cua serine, threonine, nhom guanidine cua arginine va imidasol cua tryptophan. Qua trnh ket hp enzyme se xay ra mot cach trc tiep khi chat mang co cha cac nhom chc co kha nang lien ket trc tiep noi tren trong phan t protein enzyme. V du goc anhydride maleic lien ket vi nhom -NH2 cua lysine. Trong cac trng hp khac s lien ket gia chat mang va protein enzyme ch xay ra sau khi chat mang a c hoat hoa . Viec hoat hoa s bo chat mang co the c thc hien bang nhieu cach. V du, cac nhom hydroxyl cua dextran hoac cac polyoside khac co the c hoat hoa bang bromur cyan (BrCN). Sau o chat mang a c hoat hoa mi lien ket vi enzyme (hnh 19).

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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Hnh 19. Gan enzyme vi vat mang a c hoat hoa bang bromur cyan (BrCN).

Cac chat mang co cha nhom amin nh aminobenzoylcellulose, polyaminostyrol co the c hoat hoa bang phan ng diazo. V du, polyaminostyrol trc het c diazo hoa bang natri nitrit trong moi trng acid thanh muoi diazo, sau o mi ket hp vi enzyme: R NH2
R: Goc alkyl, aryl

NaNO2/HCl

R N2+X

Muoi diazo cua chat mang X: Goc acid

N2+X

+ Enzyme

OH

RN=N

OH Enzyme

chat mang hoa

Enzyme lien ket vi chat mang cha c hoat hoa

Enzyme lien ket vi ac hoat

Phan ng ket hp enzyme c tien hanh nhanh chong trong ieu kien nhiet o thng va dung dch nc trung tnh. Neu chat mang co cha nhom amin co the hoat hoa bang cach cho tac dung vi phosgen hoac tiophosgen e tao thanh dan xuat isosianat hoac izothyosianat. Cac nhom isosianat hoac izothyosianat pH trung tnh se lien ket de dang vi nhom -NH2 cua lysine. Cl

O=C
Cl Cl

R N = C = O + HCL

R NH2 +

S =C Cl R N = C = O + H2N - E

R N = C = S + HCl R NH CO NH - E

Bang phng phap nay ngi ta a ieu che c cac dan xuat enzyme co nh nh trypsin, chimotrypsin, -, -amylase, glucoamylase. Neu chat mang co cha nhom COOH nh CM-cellulose hoac nha tong hp th can c hoat hoa trc khi gan ket vi enzyme bang cac phng phap azit, carbodiimit.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme
Hoat hoa bang phng phap azit: O
CM-cellulose

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H2N NH2
Hydrazin

O O CH2 C NH NH2
Hydrazit

- O CH2 C O CH3

NaNO2 E
nh

O - O CH2 C N = N+- NH
Azit

E NH2

O - O CH2 C NH
Enzyme co

Cac enzyme nh trypsin, DNA-ase, chimotrypsin, bromelin a c co nh bang phng phap nay. Hoat hoa bang phng phap carbodiimit: - O CH2 COOH + -OH
CM-Cellucose

N=C=N N,N-Dicyclohexylcarbodiimide

+ H2N E
Enzyme

- O CH2 CO-NH E + NH - C O NH Dixyclohexylure - OH V phan ng xay ra trong moi trng acid yeu (pH=5) nen phng phap nay ac biet thuan li oi vi cac enzyme co tnh acid nh pepsin. Ket hp ong hoa tr gia cac phan t enzyme vi chat mang. Co nh enzyme bang cach lien ket ong hoa tr c thc hien bang nhieu cach khac nhau: 1/ Lien ket ong hoa tr c tao ra gia cac nhom phan ng cua vat mang va enzyme; trong mot so trng hp vat mang can c hoat hoa s bo; 2/ Cac phan t enzyme sau khi c hap phu tren chat mang hoac nam trong long phan t chat mang lien ket vi nhau nh tnh lng chc hoac tnh a chc cua chat phan ng. Ngi ta thng dung aldehyde glutaric e gan cac nhom amin cua lysine cac phan t enzyme. V du, trypsin co nh trypsin c ieu che banh cach dung aldehyde glutaric 2% e lien ket cac phan t enzyme va gan chung vao aminoethylcellulose. b/ Phng phap goi enzyme trong khuon gel.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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e goi enzyme vao khuon gel ngi ta tien hanh trung hp hoa hoc cac gel khi co mat ong thi enzyme. Sau khi hoan thanh qua trnh trung hp enzyme b gi chac trong gel. Gel a co enzyme co the nghien nho bang cach ep qua ray co lo nho va say kho nhiet o thap. Dan xuat enzyme loai nay lan au tien do Bernfeld (1933) thu c bang cach trung hp acrylamide vi N,N-methylenbisacrylamide. Phng phap nay thng dung do chat trung hp thu c de tao thanh dang hat va tuy thuoc ieu kien tien hanh ma gel co the co o xop khac nhau. Sau ay la mot so phng phap goi enzyme. Cac gel co the c hnh thanh t cac polymer tong hp nh acrylamide, hydroxyethyl-2-metacrylate, tao phc cang cua bang ethyleneglycol-dimetacrylate, polyvinyl, polyuretan. Enzyme c hoa tan hoac phan tan trong mot dung dch monomer va sau o trung hp trong s co mat cua mot hay nhieu tac nhan tao phc cang cua. Gel co the cat thanh mang at tren mot gia the ran, hoac cung co the nghien thanh bot sau khi loai tr nc. Alginate va caraghenan lay t rong bien thng co kha nang tao gel rat tot va rat thuan li e bao goi cac enzyme hoac te bao nguyen ven. Cac enzyme cung co the b nhot trong cac lo nho cua cac si tong hp. Vi cach nay ngi ta cho dch long chay ben trong si, do o han che c s phan cc be mat va s bt lap thng gap vi cac mang. Mot nhu tng cua cellulose triacetate trong methylene chlorua va enzyme trong dung dch em co cha glycerol c ep qua mot khuon loc di ap suat nit. Cac si i ra khoi khuon c nhung vao mot cai be ong tu co cha toluel, sau o c lam kho trong chan khong. Cac si nay ben vi acid yeu hoac kiem yeu, lc ion cao va chu c mot so dung moi hu c. Tuy nhien phng phap nay ch thch hp oi vi nhng enzyme kho b mat hoat tnh trong cac dung moikhong hoa lan vi nc. Phng phap goi enzyme trong cac bao vi the (microcapsul). Enzyme c goi trong cac bao vi the co mang ban tham c tao ra t cac polymer co kch thc lo u nho e ngan can s khuech tan cua enzyme ra ngoai va u ln e c chat i vao va san pham phan ng i ra de dang. Co nhieu phng phap goi enzyme trong microcapsul. Sau ay la mot trong cac phng phap o. Cho mot dung dch loang cha enzyme va mot

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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monomer a nc trong mot dung moi hu c khong hoa lan trong nc e tao nhu tng, sau o them mot monomer k nc e gay phan ng trung hp tao ra mot mang bao quanh nhng giot long nho. Thng phai them vao mot tac nhan hoat ong be mat e lam ben nhu tng cung nh e ieu chnh cac capsul co kch thc mong muon t 1 en 100 m. Phng phap tien polymer e goi cac chat xuc tac sinh hoc. Co nh enzyme bang phng phap nay co nhng u iem nh sau: - Qua trnh goi rat n gian trong nhng ieu kien nhe nhang; - Cac chat tien trung hp khong cha cac monomer von co the gay anh hng xau en enzyme a c goi; - Cau truc li cua gel co the c ieu chnh bang cach dung cac tien polymer co chieu dai chuoi bat ky; - Cac tnh chat hoa ly cua gel (nh s can bang gia tnh hao nc va tnh k nc, ban chat ion) co the nh hng trc bang cach chon cac tien polymer thch hp von a c tong hp hoa hoc trong ieu kien vang mat enzyme. Di ay la mot v du ve phng phap tien polymer. o la phng phap tao lien ket cheo gia cac tien polymer bang cach chieu tia cc tm e goi enzyme. Khi chieu tia cc tm gan len dung dch co cha chat tien polymer va enzyme th se tao ra cac lien ket cheo gia cac goc cua tien polymer va mot gel c hnh thanh bao lay enzyme. S goi enzyme bang phng phap nay co the tien hanh ieu kien nhe nhang, tranh c cac thay oi pH qua kiem hoac qua acid, thi gian lai rat nhanh, ch trong vong t 3-5 phut. Vi phng phap nay co the goi enzyme, te bao vi sinh vat hoac cac bao quan. c/ Co nh enzyme bang phng phap hap phu tren cac chat mang co hoac khong co ien tch. Vi phng phap nay enzyme c hap phu tren cac chat co hoat tnh be mat nh than hoat tnh, cellulose, tinh bot, dextran, collagen, albumin, agarose, chitin, polyacrylamide, nilon, polystyrol v.v...va mot so nha trao oi ion, silicagen, thuy tinh. Trong trng hp chat mang khong co lo enzyme c nh vao chat mang thanh tng lp tren be mat; khi chat mang co lo th cac phan t enzyme se xam nhap vao trong cac lo. 3. Mot so ac tnh cua enzyme co nh. Viec co nh lam thay oi ang ke nhieu tnh chat cua enzyme nh o ben, tnh ac hieu, ac iem ong hoc. Thong thng viec co nh lam tang

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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ang ke o ben cua enzyme. V du, co nh protease lam tang o ben vi nhiet gap chuc nghn lan. Sau khi c co nh khong nhng o ben vi nhiet cua enzyme tang len ma pham vi nhiet o toi thch cua enzyme cung c m rong. Nhiet o cao lam cho enzyme b bien tnh, cau truc bac ba cua enzyme b pha v va enzyme b mat hoat tnh. Khi enzyme c co nh nh nhieu lien ket gan vi chat mang qua trnh bien tnh se b ngan can, lam cho enzyme chu c nhiet o cao. Trong trng hp oi vi cac enzyme thuy phan protein, v du papain va trypsin s co nh lam ngan can mot phan hoac hoan toan hien tng t phan. ieu nay rat co y ngha thc te v no giup lam tang thi gian lam viec cua enzyme. S bien oi ac iem ong hoc cua enzyme do viec co nh gay ra co the c minh hoa qua v du oi vi enzyme glutamate dehydrogenase: oi vi enzyme co nh tren mang collagen hang so Michaelis danh cho acid glutamic tang 6 lan, tac dung hoat hoa cua ADP cung tang len ang ke. Mot trong cac nguyen nhan lam bien oi tnh chat cua enzyme khi chung c co nh la s thay oi cua phan ng moi trng. Enzyme thng c co nh tren cac chat mang co ien tch. V du mot chat trao oi anion mang ien tch am c bao quanh bi mot moi trng giau proton, va v the pH trong cac lp nam ke sat vi enzyme c co nh se thap hn so vi pH cua moi trng dung dch. ieu o lam cho ng cong phu thuoc pH cua hoat tnh enzyme co nh tren cac chat mang anionit, cationit va trung tnh se khac nhau. 4. ng dung cua enzyme co nh. a/ Trong cong nghiep. - T nam 1969 Wilson a xay dng thanh cong mot xng thc nghiem e san xuat lien tuc glucose bang glucoamylase co nh; - T 1971 ngi ta a thanh cong trong viec dung chimotrypsin co nh tren carboxymethylcellulose e lam ong tu sa thay cho renin at tien; - Enzyme rasemase co nh a c s dung e chuyen toan bo Daminoacid thanh L- aminoacid tng tng, lam tang gia tr dinh dng cua san pham len gap oi. - Lam trong nc trai cay bang cach x ly enzyme phan giai pectin hay thuy phan protein trong bia bang protease hoan toan co the c thc hien trong cac cot cha cac enzyme co nh tng ng.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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- Co nh enzyme glucoisomerase trong cot hoan toan co hieu qua oi vi viec ngan nga s chuyen hoa glucose thanh fructose, lam cho o ngot cua nc giai khat b giam. Nh co nh enzyme glucoisomerase viec thu nhan xiro gluco-fructose ngay nay a tr thanh mot quy trnh cong nghiep quan trong va pho bien. o chu nhiet cao cua enzyme nay cho phep thc hien qua trnh san xuat nhiet o 60-70o, lam giam ang ke kha nang nhiem khuan cua thiet b enzyme. b/ Trong y hoc. - Urease gan trong vi tieu cau a c s dung co hieu qua e loai tr urea cua mau trong than nhan tao; - Vi tieu cau cha catalase a co the thay the mot cach co hieu qua so catalase b thieu trong c. - a vi tieu cau co gan enzyme L-asparaginase vao c the co kha nang c che s phat trien cua mot so u ac tnh v s phat trien cua nhng u nay phu thuoc vao s co mat cua L-asparagin. c/ Trong phan tch hoa sinh. Glucoamylase gan ong hoa tr vi polystyrol c dung e xac nh t ong glucose; tuc; ien cc urease co nh dung e xac nh t ong urea tren dong lien

ien cc alcoholoxydoreductase co nh c dung e xac nh methanol, ethanol trong dung dch nc. Co nh enzyme tao ra kha nang nghien cu hang loat cac van e ly thuyet cua enzyme hoc. Vi du co nh enzyme co the c s dung e ngan can s phoi hp ngau nhien cua cac phan di n v cua cac enzyme oligomer. S dung viec co nh co the giai quyet van e lieu mot enzyme dang cac phan di n v co hoat tnh hay khong. Neu cac phan di n v trang thai co nh co hoat tnh th qua so sanh hoat tnh cua enzyme vi hoat tnh cua cac oligomer co nh se thu c nhng thong tin co ga tr ve vai tro cua s tng tac gia cac phan di n v trong viec thc hien chc nang cua enzyme. Ngay nay ta biet ro rang phan ln enzyme noi bao hoat ong trong moi trng tng t nh gel hoac chung c goi trong mang ti the, luc lap hoac cac c quan t khac. V vay, cac enzyme co nh co the la mo hnh tot e nghien cu hoat ong cua enzyme. Ky thuat co nh khong ch dung cho cac che pham enzyme ma co the dung cho cac te bao vi sinh vat nguyen ven. e co nh cac te bao vi sinh vat

GS.TS. Mai Xuan Lng

Khoa Sinh hoc

Enzyme

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ngi ta thng dung gel polyacrylamide, cellulose, caraganine va cac vat lieu khac. ang lu y la trong nhieu trng hp hoat tnh enzyme cua cac te bao co nh cao hn nhieu so vi cac enzyme t do. Cac te bao co nh tham ch co the thc hien c cac qua trnh sinh hoa tinh vi va phc tap. V du cac te bao Rhisobium lupini cua not san cay lupin vang gi c rat lau hoat tnh co nh am cua chung.

GS.TS. Mai Xuan Lng

Khoa Sinh hoc