Professional Documents
Culture Documents
Enzyme
Enzyme
F7G
GIAO TRNH
ENZYME
2005
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Enzyme
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 -
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
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.
Enzyme
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Enzyme
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
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Enzyme
Biocytin
Tetrahydrofolate
Acid lipoic
CO2
Biotin
Folate
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Enzyme
1. Oxydoreductase
2. Transferase
3. Hydrolase
4. Liase
5. Isomerase
6. Ligase
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
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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Enzyme
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
Ea (Kcal/mol)
18.000
11.700
< 2.000
16.800
10.200
4.500
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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
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
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]
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Enzyme
Nhan hai ve cho [S] va bien oi phng trnh, ta co:
v =
V[S]
Km + [S]
Phng trnh
nay con cho phep
tm hieu nhieu
kha canh quan
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Enzyme
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Enzyme
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.
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Enzyme
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
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Enzyme
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
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Enzyme
E + A (EA F C) F + B (FB FD) E
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
ED
E
EB
EA
(EAB ECD)
C
EC
D
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Enzyme
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.
-Glucosidase
Urease
Trypsin
-Amylase tuyen tuy
-Amylase mam lua mach
Carboxypeptidase
Phosphatase kiem huyet
tng
Phosphatase acid huyet
tng
Arginase
C chat
Albumin trng
casein
Hemoglobin
Benzyloxycarboxylglutamyltyrosine
-Metylglucoside
Maltose
Urea
Protein
Tinh bot
Tinh bot
Cac chat khac nhau
2-Glycerophosphate
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
2-Glycerophosphate
4,5-5,0
Arginine
9,5-9-9
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.
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Enzyme
9. Anh hng cua nhiet o.
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.
- 19 -
Enzyme
V. C CHE ENZYME
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 :
COOHCCOO-
CH2
CH2
+ A
OOCCH +
AH2
- 20 -
Enzyme
COOSuccinate Chat nhan Fumarate
CH2
CH2
C=O
COOH
CH2
CH2
CH2
CH2
CH2
COOH
Oxalate
COOH
Malonate
COOH
Glutarate
CH2
CH2
COOH
COOH
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 .
Enzyme
- 21 -
- 22 -
Enzyme
EI
ES + I ESI
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
- 23 -
Enzyme
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:
[ESI]
=
Ki
[ES][I]
Va phng trnh ao ngc kep se la
1
V
1
1
[I]
Km
= x + 1 +
[S] Vmax
Ki
Vmax
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.
- 24 -
Enzyme
SO2NH2
NH2
COOH
Acid p-aminobenzoic
Sulfanilamide
Enzyme
- 25 -
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.
Enzyme
- 26 -
Trong
nhng
he
thong multienzyme nay toc
o cua mot phan ng nao
o thng xac nh toc
o cua toan bo
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
- 27 -
Enzyme
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,
Enzyme
- 28 -
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.
- 29 -
Enzyme
VIII. HE THONG CASCADE - BIEN OI ONG HOA TR.
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.
Enzyme
- 30 -
- 31 -
Enzyme
IX. HOAT HOA ENZYME.
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
- 32 -
Enzyme
X. TNG TAC PROTEIN - PROTEIN.
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
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).
- 33 -
Enzyme
XI. TNH AC HIEU CUA ENZYME OI VI C CHAT.
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
C=O
NH3+
HN
NH2+
HN
-Benzoyl-L-lysinamide
-Benzoyl-L-argininamide
C=O
C=O
NH3+
HN
-Benzoyl-L-ornitinamide
NH2+
HN
-Benzoyl-L-homoargininamide
- 34 -
Enzyme
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
NH3+
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:
Enzyme
- 35 -
- 36 -
Enzyme
-D-Glucoso-6-phosphate
Enz-O-(P) + Glucoso-1-(P).
bi
- 37 -
Enzyme
Enzyme
- 38 -
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
- 39 -
Enzyme
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
C
-RO
C
O
O
C - OH
O
+H2O
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.
- 40 -
Enzyme
H C C H
O
O
O
Ethylene-phosphate
CH3
CH3
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
- 41 -
Enzyme
CH3
CH3
C=O
H
Acetaldehyde
CH3OH
Methanol
H OCH3
Semiacetal
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:
- 43 -
Enzyme
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.
O2N
O
O - C CH3
HO C
p-Nitrophenylacetate chymotrypsin
O
O - C - CH3 + O2N
Acetyl-enzyme
OH
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:
- 44 -
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
Nhom phan ng
Papain, ficin,
glyceraldehyde phosphate
dehydrogenase
HS-CH2-CH
Cystein
Suxinic thiokinase,
glucoso-6-phosphatase
HO-CH2-CH
Serine
HO-CH2-CH
Serine
O
- O-P-O-CH2 -CH
OPhosphoryl ester
O
R-C-S-CH2 -CH
Acyl thioester
-CH2 - CH
HN
-CH2 -CH
O
-O-P-N
O-
Histidine
Aldolase, transaldolase,
pyridoxalphosphate
enzyme
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
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 .
Enzyme
- 46 -
XIII. ISOENZYME
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.
- 47 -
Enzyme
XIV. CAC NHOM ENZYME
A + E.NAD.H + H+
va AH2 + E.NADP+
A + E.NADP.H + H+
E.FH2 + A
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.
- 48 -
Enzyme
- 49 -
Enzyme
NAD+ (hay NADP+)
Enzyme
- 50 -
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)
- 52 -
Enzyme
FAD
FAD.H2
- 53 -
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
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.
- 54 -
Enzyme
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
Sedoheptuloso-7phosphate:glyceraldehyde Transcetolase
-3-phosphate
glycolaldehyde transferase
2.4.1.1
1,4--DGlucan:ortophosphate-D-glucosyltransferase
2.4.1.1
3
UDPlgucose:D-fructose
2--Dglucosyltransferase
L-Aspartate:2cetyoglutarate
aminotransferase
Sacchrososyntase
2.6.1.5
L-Tyrosine:2cetoglutarate
aminotransferase
Tyrosinotransferase
2.7.1.1
ATP : D-Hexoso-6phosphotransferase
Hexokinase
2.6.1.1
Phosphorylase
Aspartate
aminotransferase
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
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.
- 55 -
Enzyme
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.
Enzyme
- 56 -
- 57 -
Enzyme
Ten enzyme
Phan ng
- 58 -
Enzyme
4.1.1.1
Pyruvate decarbocylase
D-Ribuloso-1,5-diphosphate +
CO2 +H2O = 2,3-phospho-Dglycerate
D-Fructoso-1,6-diphosphate =
D-Dioxyacetonephosphate +
D-Glyceraldehyde-3-phosphate
4.2.1.2
4.3.1.1
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.
- 59 -
Enzyme
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 +
Enzyme
- 60 -
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
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
Enzyme
- 63 -
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.
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.
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Enzyme
XVI. S DUNG ENZYME TRONG CONG NGHE SINH HOC.
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
Enzyme
- 66 -
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
Enzyme
- 67 -
Enzyme
- 68 -
- 69 -
Enzyme
- 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;
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).
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Enzyme
Hnh 19. Gan enzyme vi vat mang a c hoat hoa bang bromur cyan (BrCN).
NaNO2/HCl
R N2+X
N2+X
+ Enzyme
OH
OH
RN=N
Enzyme
chat mang
hoa
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
R NH2 +
R N = C = O + HCL
Cl
Cl
S =C
R N = C = S + HCl
Cl
R N = C = O + H2N - E
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.
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Enzyme
-
- O CH2 C O CH3
CM-cellulose
NaNO2
O CH2 C NH NH2
Hydrazin
O
E
O
Hydrazit
E NH2
- O CH2 C N = N+- NH
O
- O CH2 C NH
Azit
Enzyme co
nh
N=C=N -
+ H2N E
N,N-Dicyclohexylcarbodiimide
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.
- 73 -
Enzyme
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
- 74 -
Enzyme
Enzyme
- 75 -
Enzyme
- 76 -
Enzyme
- 77 -