“The reaction ‘mechanism Sec. 72 Enzymate Reaction Fundamentals 397 There are six classes of enzymes and only six: 1. Oxidoreductases AH, +B+E A+ BH; +E 2. Transferases AB+C+E3AC+B+E 3. Hydrolases AB+H,O +E AH + BOH+E 4. Isomerases A+Eiso=A+E 3. Lyases AB+E>A+B+E 6. Ligases A+B+ESAB+E ‘More information about enzymes can be found on the following two web sites: hup:/tus.expasy.org/enzyme/ and www.chemgmw.ac.ub/iubmblensyme ‘These sites also give information about enzymatic reactions in general 7.22 Mechanisms In developing some of the elementary principles of the kinetics of enzyme rections, we shall discuss an enzymatic reaction that has been suggested by Levine and LaCourse as part of a system that would reduce the size of an arti ficial kidney.* The desired result is the production of an antficial kidney that ‘could be wom by the patient and would incorporate a replaceable unit for the climination ofthe nitrogenous waste products such as uric acid and creatinine {In the microencapsulation scheme proposed by Levine and LaCourse, the enzyme urease would be used in the removal of urea from the bloodstream, Here, the catalytic action of urease would cause urea to decompose into ammonia and carbon dioxide. The mechanism of the reaction is believed to proceed by the following sequence of elementary reactions: 1. The enzyme urease (E) reacts with the substrate urea (S) to form an enzyme-substrate complex (E * S) NH,CONH, + Urease — [NH,CONH,*Urease]* (7-13) . This complex (E + S) can decompose back to urea (S) and urease (E) INH,CONH, * Urease]” —2> Unease + NH;CONH; (7-14) 3. Or it can react with water (W) to give the products (P) ammonia and carbon dioxide, and recover the enzyme urease (E). INH,CONH + Urensel" + H,0 24 2NH + CO, + Urease (7-15) We see that some ofthe enzyme ade! othe solution binds to the urea and some remains unbound. Although we can easily measure the total concentration of enzyme, (E,), itis difficult to measure the concentration of free enzyme, (E). ®N. Levine and W. C. LaCourse, J. Biomed, Mater. Res. 1, 275 (1967).We need to replace ‘bound eneyme ‘concentration (E) it ‘he ate ln 398 Fescion Mechansms, Pamways, Bloreactions, and Boreacios Chap. 7 Letting E, S, W, E'S, and P represent the enzyme, substrate, water, the enzyme-substrate complex, and the reaction products, respectively, we can write Reactions (7-13), (7-14), and (7-15) symbolically in the forms S+E—44 Bes (7-16) Eves 44 B+s 17) EeS+W 44 P+E 7-18) Here P = 2NH; + CO; ‘The corresponding rate laws for Reaction (7-16), (7-17), and (7-18) are ris=—hy (EMS) (7-164) E+S) (-iTA) 3 (E +S) (W) (7-184) The net rate of disappearance of the substrate, (EMS) — k(E + S) (7-19) ‘This rate law is of not much use to us in making reaction engineering calculations because we cannot measure the concentration of enzyme substrate ‘complex (E + S). We will use the PSSH to express (E + S) in terms of measured variables. The net rate of formation of the enzyme-substrate complex is es = R(EMS)— h(E + 8) — ky(WE* S) 7-20) =r Using the PSSH, rp.s = 0, we solve equation (7-20) for (E + S) K(E\S) ES) RW) aa and substitute for (E + S) into [Equation (7-19)] (EMS) +k(W) k(EMS)~, = HALENSIOW) Ky s(W) ued We still cannot use this rate law because we cannot measure the unbound enzyme concentration (E); however, we can measure the total enzyme concen tration, E,.“Toa enzyme con- centration = Bound ++ Free enzyme con- ‘Tumover umber ‘S0c.7.2 Enzymatic Reaction Fundamentals 309 In the absence of enzyme denaturization, the total concentration of the enzyme in the system, (E,), is constant and equal to the sum of the concentra- tions of the free or unbonded enzyme, (E), and the enzyme-substrate complex, (E+): €)=@+E-s) (7-23) Substituting for (E + ) A(ENS) @)=©+ Ea solving for (E) (Ekg + kW) ©) EF RIW)+ hiS) substituting for (E) in Equation (7-22), the rate law for substrate consumption is Fi kWHENS) aE (7-24) RS) +R FKOW) Note: Throughout, E, = (E,) = total concentration of enzyme with typical units (mol or g/d) 7.2.8 Michaelis-Menten Equation Because the reaction of urea and urease is carried out in aqueous solution, water is, of course, in excess, and the concentration of water is therefore con- sidered constant. Let fag = Ky(W) and Ky = 4 Dividing the numerator and denominator of Equation (7-24) by &y, we obtain a form of the Michaelis-Menten equation: (7-25) The parameter hy is also referred to as the turnover number: tis the number of substrate molecules converted t0 product in a given time on a igle-enzyme molecule when the enzyme is saturated with substrate (.c. all the active sites on the enzyme are occupied, S>>Ky). For example, tumover number for the decomposition H:0; by the enzyme catalase is 40 x 108s" ‘That is, 40 million molecules of HO. are decomposed every second on & single-enzyme molecule saturated with Hy03. The constant Ky, (moliém’) is called the Michaelis constant and for simple systems is a measure of theMichaelis constant Kee 400 Reaction Mechanisms, Pathways, reactions, and Bioreacors Cha attraction of the enzyme for its substrate. so it’s also called the afity ¢ sant. The Michaelis constant, Ky. for the decomposition of H,0; discus ‘earlier is 1.1. M while that for chymotrypsin is 0.1 M? If, in addition, we let Vag represent the maximum rate of reaction fe sgiven total enzyme concentration, Vax = Kea E,) de Mictuetis-Menten equation takes the familiar form = Font) Kt®) =r a. For a given enzyme concentration, a sketch of the rate of disappearance of substrate is shown as a function of the substrate concentration in Figure 7. Figure 7-6 Michaci-Menten plot identifying the parameters Vay 3nd Ks A plot of this type is sometimes called a Michaelis strate concentration, Kyy > (S), ‘Menten plot. At low s Vona(S) Ky and the reaction is apparent first order in the substrate concentration. At h substrate concentrations, “n= (> Ky and the reaction is apparent zero order 15> Va "D. L. Nelson and M. M. Cox, Lehninger Principles of Biochemistry, 34 od. (% ‘York: Worth Publishers, 2000),Imerpretation of Michacis constant ‘Sec.7.2 Enzymatic Reaction Fundamentals, 401 Consider the case when the substrate concentration rate is equal 10 one-half the maximum rate, then Fat Sa) 7 =o 7-27) Solving Equation (7-27) for the Michaelis constant yields a= Sid (7-28) ‘The Michaelis constant is equal tothe substrate concentration at which the rate of reaction is equal to one-half the maximum rate. The parameters Vaus and Ky characterize the enzymatic reactions that are described by Michaelis-Menten kinetics. Vpas is dependent on total enzyme concentration, whereas Ky is nat. ‘hwo enzymes may have the same valUes Tor Kx Dut have aiterent reac- tion rates because of different values of Ky. One way to compare the catalytic efficiencies of different enzymes is to compare the ratio Key/Ky. When this ratio approaches 108 to 10? (dm’/molls) the reaction rate approaches becoming diffusion-limited. That is, it takes a long time for the enzyme and substrate 0 find each other, bt once they do they react immediately. We will discuss dif- fusion-limited reactions in Chapters 11 and 12. Example 7-3 Evaluation of Michaelis-Menten Parameters Vy. and Kyy Determine the Michaelis-Menten parameters Vy, and Ky for the reaction Urea + Urease ==? (Urea-Urease!’ > 2NH, + CO; + Urease ‘The rate of reaction is given asa function of wea concentration in this table Comino’) | 02002 001 0005 0002 rua knollmi-a | 108088 038-02 009 Solution Inverting Equation (7-26) gves us = Ky Fan) BEENLineweaver-Burk pe For emymatic reactions, the two ey rate tow puraeters are Ve 308 Kr 402 Reaction Mechanisms, Pathways, Sloreactons, and Biereacors E7-3.1 in the form of a Lineweaver-Burk plot. The intercept is 0.75. so = 0.75 m?-s/kmol Raw Axo Processe DATA Figure E71. (s)Michacis-Menten pot (b) Lineweaver-Burk plo ‘Therefore, the maximum rate of reaction is Vag = 133 kmolfmn-s 1.33 mol/dm?-s PY = slope = 0025 x, = 0.0266 kmol/m? chap. 7 67-32) {A plot ofthe reciprocal reaction rate versus the reciprocal urea concentration should be a straight line with an imercept 1/Vny, and slope Ky/ Vis. This type of plot is called a Lineweaver-Burk plot. The data in Table E7-3.1 are presented in Figure j mien Vere tinal’) nets) mow om ve 30 ass om oss 500 ue ‘or 038 10 26 tos 02 2000 so ‘02 10 5000 ut ss ‘ See it | = oS From the slope, which is 0.02 s, we can calculate the Michaelis constant, Kyanes Wooit plot ‘S0c.72 Enzymatic Reaction Fundamentals 403 Substituting Ky and Vqa, into Equation (7-26) T3Cwe 0266+ Con where Cg has units of kmol/m? and —r, has units of kmol/m’:s. Levine and LaCourse suggest thatthe ttal concentration of urease, (E,), corresponding to the value of Vas above is approximately 5 g/dm. In addition to the Lineweaver-Burk plot, one can also use a Hanes-Woolt pilot or an Eadie-Hofstee plot. Here $= Cig. td =Fy © iy Equation (7-26) (733) © 7-26) nS) vey can be eaanged inthe follwing fom. For he Eae-Hofstee form. aaa i = af) was) Fer the Hanee- Wolf form, ws have (E7-3.5) For the Eadie-Hofstee model we plot rg a8 a function of (-r/S) and for the Hanes-Woolf model. we plot [(S)/-r5} a8 a function of (S). The EadieHofstee plot ‘does not bias the points at low substrated concentrations, while the Hanes-Woolf plot gives » more accurate evaluation of Vz. In Table E7-32, we add two columns {o Table E7-3.1 10 generate these plots (Coe, = 5). ‘Tame E72. RaW AND PaocesstD Dara > 5 1 Te oo, =e Gkrolim!) (kmovim' +s) _(mkwel)_(m+ Amol) _—_) ais, 030 Toe 30 O93 ons 3a 00 03s 500 Le ome ms aot er) 263 ames 38 000s 0202000 500 amso 40 002 009 000 nat oom as Plotting the data in Table E7-3.2, we arrive at Figures E7-3.2 and E7-3.3. -—_____, a 7 37 | 4 | |) Figure £7.32 Hanes-weot plo Figure £7.33 Esdie- Hote plot404 Reaction Mechanisms, Pathways, Bloreactons, and loraciors Cha Regression Equation (7-26) was used in the egression program of Polymath withthe follow results for Vanax and Ky ‘Nonlinear regression (L-M) Model: rate = Vmax" Cureay(Km+Curea) Variable © Ini. quess _Value v4 ‘vane a 12087502 -0,0898303 Ka 0.02 00233322 0.003295 ‘Nontinear regression settings Max # iterations = 64 Precision Ra = 0.999061 Re2adj = 0.9987481 Raed = 0,0047604 | variance = 1.888604 The Product-Enzyme Complex In many reactions the enzyme and product complex (E + P) is formed direc from the enzyme substrate complex (E + S) according to the sequence E+S <=? EoS =? EoP == P+S Applying the PSSH to both (E + S) and (E * P), we obtain ‘Briggy-Haldane Rae Law =p, = Vaal Cs CoKe) aa C5 + Rea + KeCe which is often referred to as the Briges-Haldane Equation (see Problem P7-1 and the application of the PSSH to enzyme kinetics often called t Briggs-Haldane approximation, 7.2.4 Batch Reactor Calculations for Enzyme Reactions ‘A mole balance on urea in the batch reactor gives aN, ‘Mole balance a ree v Because this reaction is liquid phase, the mole balance can be put in the fe lowing form: Maes a a3Rate hw Combine Imegeate Time to achieve a comerion ti 4 ‘teh enzymatic ‘Sec. 7.2 Enaymalie Reaction Fundamentals 405, ‘The rate law for urea decomposition is ates aan att Coren Substituting Equation (7-31) into Equation (7-30) and then rearranging and integrating, we get (732) ‘We can write Equation (7-31) in terms of conversion as Crea = Cons] = 2) 732) ‘The parameters Ky, and Vin. can readily be determined from batch reactor data by using the integral method of analysis. Dividing both sides of Equation (7-32) by #K/Vigs and cearranging yields We see that Ky and Vg an be determined from the slope and intercept of a plot of i/t In{i/(1— X0] "versus X/t. We could also express the Michaelis-Menten equation in terms of the substrate concentration S: 33) where S, is the initial concentration of substrate. In cases similar to Equation (7-33) where there is no possibility of confusion, we shall not bother 10 enclose the substrate or other species in parentheses to represent concentration [ie., Cy = (S) = S]. The corresponding plot in terms of substrate concentra- mn is shown in Figure 7-8.408 Reaction Mechanisms, Pathways, Bloreactions, and Bloraciors Chap. 7 Figure 7-7. Evaluating Van, Example 7-4 Batch Enzymatic Reactors Calculate the time needed to convert 9% of the urea to ammonia and carbon diox- {de in a 0.5-dm* batch reactor. The intial concentration of urea is 0.1 mol/dm*, and the urease concentration is 0.001 g/dm’. The reaction is to be cartied out isother rally atthe same temperature at which the data in Table E7-3.2 were obtained. Solution We ean use Equation (7-32), Goro 2) a 32) where Ky = 0.0266 mol/dm, X = 0.99, and Cyan = 0.1 mol/dm, Vag, Was 1.33 :mol/dims. However, forthe conditions inthe batch reactor, the enzyme eoncentra- tion is only 0.001 g/ém compared with 5 g in Example 7-3. Because Vay = Ehsy Vy forthe second enzyme concentration is 2.001 5 1.33 = 2.66 x 10-* mol/s- dm? 10266 moViim® and X Substituting into Equation (7~ 99 px 256%10* mode? a(t) (0.1 movide’y(0.99) 2.66% 10"* mot/ém’7s 0.01)" 2.66% 10~* molds = 460 5+ 380s = 840 $14 minutes)Sec.7.2 Eraymale Reason Fundamentais 407 Effect of Temperature ‘The effect of temperature on enzymatic reactions is very complex. If the ‘enzyme structure would remain unchanged as the temperature is increased, the rate would probably follow the Amthenius temperature dependence. However. as the temperature increases, the enzyme can unfold and/or become denatured and lose its catalytic activity. Consequently, a the temperature increases, the reaction rate, ~rs,inereases up to a maximum with increasing temperature and then decreases as the temperature is increased further. The descending part of this curve is called temperature inactivation or thermal denatorizing.'" Figure 7-9 shows an example of this optimum in enzyme activity." 29 40 31 32 a3 94 a5 96 a7 a8 sr a) x08 Figure 78 Catalytic breakdown rae of H:0; depending on temperature. Coutesy ofS Aiba. AE. Humphrey. and N-F Mill, Blachemical Engineering, Acari Press (1973), “ML L. Shuler and F. Karp, Bioprocess Engineering Basic Concepts, 2nd ed. (Upper Saddle River. N..: Prentice Hall, 2002), p. 77 "¥s, Aiba, A. E, Humphrey, and N. F. Mills, Biochemical Engineering (New York Academic Press, 1973), p. 47.408 Reaction Mechanisms, Patiways, Soreactons. and Bioreactors Ci Side note: Lab-on-a-chip. Enzyme-catalyzed polymerization of nucleot is a key step in DNA identification, The microfluidic device shown in | lure SN7.1 is used to identify DNA strands. It was developed by Profe: Mark Burns's group at the University of Michigan, il eo eee Ee 11 ime INT MN essait igure SN7.1Microfuidic device to identity DNA. Coustesy of Science, 282, 484 (1995). {In order to identify the DNA, its concentration must be raised to a level t can be easily quantified. This increase is typically accomplished by replic ing the DNA in the following manner. After a biological sample (e.g. pt fied saliva, blood) is injected into the micro device, itis heated and 1 hydrogen bonds connecting the DNA strands are broken. After breaking primer attaches to the DNA to form a DNA primer complex, DNA*. enzyme ©) then attaches to this pait forming the DNA® enzyine compl DNA® + E. Once this complex is formed a polymerization reaction occt as nucleotides (ANTPs—dATP, dGTP, dCTP, and dTTP—N) attach to t primer one molecule ata time as shown in Figure SN7.2. The enzyme int acts with the DNA strand to add the proper nucleotide in the proper ord ‘The addition continues as the enzyme moves down the strand attaching ¢ rucleotides until the other end of the DNA strand is reached. At this poi the enzyme drops off the strand and a duplicate, double-stranded DNA m« ecule is formed. The reaction sequence is NA NAY OO ae SE erty st — _ OMA Sra Wa Seana ‘3 rime Somolex ~ 6 : ~~ ep © A_ + 2@_ ‘MR Ene ‘ompion He AHN gets Mawr = Figure SN7.2. Replication sequence.‘302,73 Inhiion of Eneyme Reactons 409 ‘The schematic in Figure SN7.2 can be written in terms of single-step reac- tions where N is one of the four nucleotides. ‘Complex Formation: DNA + Primer > DNA* DNA*+E ==? DNA*+E Nucleotide addition/pot}merization DNA*+E+N— DNA*+N,+E DNA*+N,+E+N-— DNA** Nz +E. ‘The process then continues much like a zipper as the enzyme moves along, the strand to add more nucleotides to extend the primer. The addition of the last nucleotide is DNA* +N, *E+N>DNAt*Nj+E where i is the number of nucleotide molecules on the original DNA minus the nucleotides in the primer. Once a complete double-stranded DNA is formed, the polymerization stops, the enzyme drops off, and separation occurs. DNA*+N,+B->2DNA+E Here 2DNA strands really represents one double-stranded DNA helix, ‘Once replicated in the device, the length of the DNA molecules can be analyzed by electrophoresis to indicate relevant genetic information. 7.3. Inhibition of Enzyme Reactions In addition to temperature and solution pH, another factor that greatly influ- ences the rates of enzyme-catalyzed reactions is the presence of an inhibitor. Inhibitors are species that interact with enzymes and render the enzyme inef- fective to catalyze its specific reaction. The most dramatic consequences of enzyme inhibition are found in living organisms where the inhibition of any particular enzyme involved in a primary metabolic pathway will render the entire pathway inoperative, resulting in either serious damage or death of the organism. For example, the inhibition of a single enzyme, cytochrome oxidase, by cyanide will cause the aerobic oxidation process to stop; death occurs in & very few minutes. There are also beneficial inhibitors such as the ones used in the treatment of leukemia and other neoplastic diseases. Aspirin inhibits the enzyme that catalyzes the synthesis of prostaglandin involved in the pain-pro- ducing process ‘The three most common types of reversible inhibition occurring in enzy- matic reactions are competitive, uncompetitive, and noncompetitive. The enzyme molecule is analogous to a heterogeneous catalytic surface in that it contains active sites, When competitive inhibition occurs. the substrate and410 Reaction Mechanisms, Pathways, Bloreactons, snd Biteactors Chap. 7 inhibitor are usually similar molecules that compete for the same site on the enzyme. Uncompettive inhibition occurs when the inhibitor deactivates the enzyme-substrate complex, sometimes by attaching itself to both the substrate and enzyme molecules of the complex. Noncompetitive inhibition occurs with enzymes containing at least two different types of sites. The substrate attaches ‘only t0 one type of site, and the inhibitor attaches only to the other to render the enzyme inactive. 7.3.1. Competitive Inhibition Competitive inhibition is of particular importance in pharmacokinetics (drug therapy). If patient were administered two or more drugs that react simults- neously within the body with a common enzyme, cofactor, or active species, this interaction could lead to competitive inhibition in the formation of the respective metabolites and produce serious consequences, Jn competitive inhibition another substance, 1, competes with the sub- strate for the enzyme molecules to form an inhibitor-enzyme complex, 2s shown here. commie Reston Steps Competitive Inhibition Pathway sninon obo fies ee ie yg cine # () +S —4 Bes o-0- i @) Bes ES E+S Co i (3) Bes 24 P+E e (4) 1+E 4 Eel (inactive) (5) Eel 4+ E+! DL. Netton and M.M, Cox, Lehninger Principles of Bochemsiry. 3 e. (New ‘York: Worth Publishers, 3000), p. 256. In addition to the three Michaelis-Menten reaction steps, there are 10 additional steps as the inhibitor reversely ties up the enzyme as shown in reac- tion steps 4 and 5, The rate law for the forthation of product is the same fef. Equation (7-18A)] as it was before in the absence of inhibitor s(E+S)‘800.73 inhiien ot Eneyme Reactons ay Applying the PSSH, the net rate of reaction of the enzyme-substrate complex Pe-s=O= hy (EMS) —ky(E + S) ~ ky (E*S) (7-35) ‘The net rate of reaction of inhibitor-substrate complex is also zer0 rent = 0 ky EMD ~ RED, (736) ‘The total enzyme concentration is the sum of the bound and unbound ‘enzyme concentrations E=E+(E+S)+ (E+) 737) Combining Equations (7-35), (7-36), and (7-37) and solving for (E + $) ‘and substituting in Equation (7-34) and simplifying rp =rg = — Lat) (7-38) Rate aw for s+K.(1 +) ‘compat K, ‘mise Vaux and Ky are the same as before when no inhibitor is present, that is, By letting Koy = Ky(1 + /K)), we can see that the effect of a competitive inhibition is to increase the “apparent” Michaelis constant, Kw. A consequence of the larger “apparent” Michaelis constant Ky is that a larger substrate con- centration is needed for the rate of substrate decomposition, —, to reach half its maximum rate Rearranging in order to generate a Lineweaver-Burk plot, Teer From the Lineweaver-Burk plot (Figure 7-10), we see that as the inhibitor (1) concentration is increased the slope increases (i.e., the rate decreases) while the intercept remains fixed.412 Reaction Mechanisms, Paways, Bloreacions, and Bloreactors Cr Increasing inibtor Concentration) [7 competi orioton No laittton Figure 7-10 Linewsaver-Burk pot for competitive inhibition. Side note: Methanol Poisoning. An interesting and important example ‘competitive substrate inhibition is the enzyme alcohol dehydrogenase (AE inthe presence of ethanol and methanol. Ifa person ingests methanol, Al will conver: it to formaldehyde and then formate, which causes blindne Consequently, the treatment involves intravenously injecting ethanol (wh is metabolized ata slower rate than methanol) ata controlled rate to tie [ADH to slow the metabolism of methanol-to-formaldchyde-to-formate sot the kidneys have time to filter out the methanol which is then excreted in ‘urine. With this treatment, blindness is avoided. For more on the met noVethanol competitive inhibition, see Problem P7-25c. 7.8.2 Uncompetitive Inhibition Here the inhibitor has no affinity for the enzyme itself and thus does not ¢ pete with the substrate for the enzyme: instead it ties up the enzyme-subs complex by forming an inhibitor-enzyme-substrate complex, (I'* E+ S) w is inactive. In uncompetitive inhibition, the inhibitor reversibly ties enzyme-substrate complex after it has been formed. ‘As with competitive inhibition, two additional react the Michaelis-Menten kinetics for uncompetitive inhibition as shown tion steps 4 and 5. steps are addScc.73 inion ol Enzyme Reactors 413 Besta Shs Uncompetiive Pathway ocsipai Pipers semicon (I) ES —2 4 BS pee : Q) BS ty E45 os as L (@) Bes hs P+E C*+0--G (4) [+EeS —+> [Ee (inactive) (5) Tees 24 13s Rate foe Starting with equation for rate of formation of product, Equation (7-34) wncompettne and then applying the psevdo-steady-state hypothesis 10 the intermediate (I+E+S), we arrive atthe rate law for uncompetitive inhibition teal week] a4 AK 1 (1 4D) ceeeB] ow ‘The Lineweaver-Burk plot is shown in Figure 7-11 for different concentrations. The slope (Ky/Viae) mains the same as the inhibition (0) con- centration is increased, while the intercept (J + (I/K)) increases, Increasing nhibter Concentration Urconpete non os ‘No Inhibition Figure 7-11 Lineweaver-Buk pot for uacompetitive inhibition,Mixed inhibition A Summary Notes Rate law for noncompetitive inkbition a4 Reaction Mechensms, Pathways, Sloreacions, and Biorssctors Chap. 7 7.33 Noncompetitive Inhibition (Mixed Inhibition)t In noncompetitive inhibition, also called mixed inhibition, the substrate and tor molecules react with different types of sites on the enzyme molecule, ‘Whenever the inhibitor is attached tothe enzyme iti inactive and cannot form products. Consequently the deactivating complex (I+ E + S) can be formed by {wo reversible reaction paths. 1. After a substrate molecule attaches to the enzyme molecule at the Subsirate site, die inhibitor molecule anaches 10 the enzyme atthe inhibitor sie 2. After an inhibitor molecule ataches to the enzyme molecule at the imibitor site, the substrate molecule attaches to the enzyme at the substrate site ‘These paths, along with the formation of the product, P, are shown here. In noncompetitive inhibition, the enzyme can be tied up in its inactive form either before or after forming the enzyme substrate complex as shown in steps 2, 3, and 4 Reaction Steps ‘Noncompetitive Pathway () B+S = Bes Active Q) E+1 H+ IE inactive) @) I+ E+S = 1+E +S (inactivey (4) S+I+E = 1+E +8 (inactive) G) E-s —+ P Again stating with the rate law forthe rate of formation of product and then applying the PSSH to the complexes (I+ E) and (I= E = $) we ave at the rate law for the noncompetitive inhibition a4) (8) +k 1+) ‘The derivation of the rate law is given in the Summary Notes on the we? and CD-ROM. Equation (7-42) is in the form of the rate law that is given for tan enzymatic reaction exhibiting noncompetitive inhibition. Heavy metal i098 such as Pb"*, Ag’, and Hg*, as well as inhibitors that react with the enzyme to form chemical derivatives, are typical examples of noncompetitive inhibitors "Im some texts, mixed inhibition is a combination of competitive and uncompetitive inhibitionSec. 73 nhibton of Enzyme Reactons a5 Rearranging : 14) el +2) 7-43) JX a No biton z 3) may ere For noncompetitive inhibition, we see in Figure 7-12 that both the slope ale) (elt+) ome wt Key D)) and imercept (fi +12)) increase with incressin Got 2 cept (F=[1+ {2 ]) increase with ing inhibitor concentration. In practice, uncompetitive inhibition and mixed inhibi tion are observed only for enzymes with two oF more substrates, Sy and S. The three types of inhibition are compared with a regtion in which no ibitors are present on the Lineweaver-Burk plot shown in Figure 7-13. Issepoalles ek aipoos Bese Gea ser eee ae geres eee No nition rie Figure 7-13 Lineweaver-Burk plots for three types of enzyme inhibition. ce416 Reaction Mechanism, Pathways, Bloreactons, and Boreaciors In summary we observe the following trends and relationships: 1. In competitive inhibition the slope increases with increasing inhi ‘concentration, while the intercept remains fixed 2. In uncompetitive inhibition, the y-intercept increases with incre: inhibitor concentration while the slope remains fixed, 3. In noncompetitive inhibition (mixed inhibition), both the y-inte and slope will increase with increasing inhibitor concentration. Problem P7-14 asks you to find the type of inhibition for the enzyme cata reaction of starch, 7.3.4. Substrate Inhibition {In a number of cases, the substrate itself can act as a inhibitor, Inthe ¢@ luncompetitive inhibition, the inactive molecules (S + E + S) is formed b reaction S+E+S —~+ S+E+S (inactive) Consequently we see that by replacing (1) by (S) in Equation (7-40) thy law for =r, is 8 Kyts+S Ky We see that at low substrate concentrations then VS and the rate increases linearly with inereasing substrate concentration. ‘At high substrate concentrations (S? / K;) >>(Ky + $), then = Fowk Ss ‘and we see that the rate decreases as the substrate concentration inc: Consequently, the rate of reaction gives through a maximum in the su concentration as shown in Figure 7-14, We also see there is an optimut ‘irate concententinn at which to operate. This maximum is found by taki etivative of Equation (7-44) wet S, to obtain org Sax = fakersubstrate inhibition S Summary Notes Sec. 73 inhibin of Enzyme Reactions a7 Figure 7-14 Suitrate reaction cae asa function of substrate concentration for subsite inhibition When substrate inhibition is possible, a semibatch reactor called a fed batch is often used as a CSTR to maximize the reaction rate and conversion. 7.35 Multiple Enzyme and Substrate Systems In the preceding section, we discussed how the addition of a second substrate, 1, to enzyme-catalyzed reactions could deactivate the enzyme and greatly inhibit the reaction, In the present section, we look not only at systems in which the addition of a second substrate is necessary to activate the enzyme, but also at other multiple-enzyme and multiple-substrate systems in which cyclic regeneration of the activated enzyme occurs. Cell growth on multiple substrates is given in the Summary Notes. Enzyme Regeneration. The first example considered is the oxidation of glu- ccose (S,) with the aid of the enzyme glucose oxidase (represented as either GO. or [E, |) to give B-gluconolactone (P): Glucose + G.0. => (Glucose : G.0.) = (B-gluconolactone - GO.) = b-sluconolactone + G.O.Hy In this reaction, the reduced form of glucose oxidase (G.O.H;), which will be represented by E,, cannot catalyze further reactions until itis oxidized back to E,. This oxidation is usually carried out by adding molecular oxygen to the system so that glucose oxidase, E,., is regenerated. Hydrogen peroxide is also produced in this oxidation regeneration step: GOH; +0, —> Go.+4, Overall, the reaction is written Glucose +0, > Hy one + 8-Gluconolactone