2 Структура и функция белка (1160071), страница 34
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Each enzyme has optimum valuesof kcat and Km that reflect the cellular environment, the concentrationof substrate normally encountered in vivo by the enzyme, and thechemistry of the reaction being catalyzed.Comparison of the catalytic efficiency of different enzymes requires the selection of a suitable parameter. The constant kcat is notentirely satisfactory. Two enzymes catalyzing different reactions mayhave the same kcat (turnover number), yet the rates of the uncatalyzedreactions may be different and thus the rate enhancement broughtabout by the enzymes may differ greatly.
Also, kcat reflects the proper-217Catalysisties of an enzyme when it is saturated with substrate, and is less usefulat low [S]. The constant Km is also unsatisfactory by itself. As shown byEquation 8-23, Km must have some relationship to the normal [S]found in the cell. An enzyme that acts on a substrate present at a verylow concentration in the cell will tend to have a lower Km than anenzyme that acts on a substrate that is normally abundant.The most useful parameter for a discussion of catalytic efficiency isone that includes both kcat and Km. When [S] <^ Km, Equation 8-26reduces to the formVo = ^ - [ E t ] [ S ](8-27)Vo in this case depends on the concentration of two reactants, E t and S;therefore this is a second-order rate law and the constant kcat/Km is asecond-order rate constant.
The factor k^JK^ is generally the bestkinetic parameter to use in comparisons of catalytic efficiency. There isan upper limit to kcat/Km9 imposed by the rate at which E and S candiffuse together in an aqueous solution. This diffusion-controlled limitis 108 to 109 M~ 1 S" 1 , and many enzymes have a value of kcat/Km nearthis range (Table 8-8).Table 8-8 Enzymes for which kCSLt/Km is close to the difiusioncontrolled limit (108 to 10 9 M-V r )^catEnzymeSubstrateAcetylcholinesteraseAcetylcholine(M)Carbonic anhydraseco 2CatalaseHCO3H2O2CrotonaseCrotonyl-CoA1.4 x 1049 x lO- 51.6 x 10861 x 104 x 10521.2 x lO2.6 x lO- 28.3 x 1071.5 x 1074 x 1074xlO75.7 x 1031.12 x lO- 528 x 109 x 10265 x 102.5 x lO- 51.6 x 1083.6 x 1072.8 x 108FumaraseFumarateMai ateTriose phosphateisomeraseGlyceraldehyde-3phosphate4.3 x 1034.7 x 10~42.4 x 108jS-LactamaseBenzylpenicillin2.0 x 1032 x lO- 5lxlO8Source: From Fersht, A.
(1985) Enzyme Structure and Mechanism, p. 152, W.H. Freeman andCompany, New York.Many Enzymes Catalyze ReactionsInvolving Two or More SubstratesWe have seen how [S] affects the rate of a simple enzyme reaction(S —» P) in which there is only one substrate molecule. In many enzymatic reactions, however, two (or even more) different substrate molecules bind to the enzyme and participate in the reaction. For example,in the reaction catalyzed by hexokinase, ATP and glucose are the substrate molecules, and ADP and glucose-6-phosphate the products:ATP + glucose> ADP + glucose-6-phosphateThe rates of such bisubstrate reactions can also be analyzed by theMichaelis-Menten approach. Hexokinase has a characteristic Km foreach of its two substrates (Table 8-6).Enzyme reaction involving a ternary complexChapter 8 Enzymes219Random orderEOrderedE + Pj + P2ESiS2S2(a)Enzyme reaction in which no ternary complex is formedFigure 8—13 Common mechanisms for enzymecatalyzed bisubstrate reactions.
In (a) the enzymeand both substrates come together to form a ternary complex. In ordered binding, substrate 1 mustbe bound before substrate 2 can bind productively.In (b) an enzyme-substrate complex forms, a product leaves the complex, the altered enzyme forms asecond complex with another substrate molecule,and the second product leaves, regenerating theenzyme. Substrate 1 may transfer a functionalgroup to the enzyme (forming E'), which is subsequently transferred to substrate 2. This is a pingpong or double-displacement mechanism.(b)Enzymatic reactions in which there are two substrates (bisubstrate reactions) usually involve transfer of an atom or a functionalgroup from one substrate to the other.
Such reactions proceed by one ofseveral different pathways. In some cases, both substrates are boundto the enzyme at the same time at some point in the course of thereaction, forming a ternary complex (Fig. 8-13a). Such a complex canbe formed by substrates binding in a random sequence or in a specificorder. No ternary complex is formed when the first substrate is converted to product and dissociates before the second substrate binds. Anexample of this is the ping-pong or double-displacement mechanism(Fig. 8-13b).
Steady-state kinetics can often help distinguish amongthese possibilities (Fig. 8-14).IncreasingPre-Steady State Kinetics Can Provide Evidencefor Specific Reaction StepsWe have introduced kinetics as a set of methods used to study the stepsin an enzymatic reaction, but have also outlined the limitations of themost common kinetic parameters in providing such information.
Thetwo most important experimental parameters provided by steady-statekinetics are kcat and kcat/Km. Variation in these parameters withchanges in pH or temperature can sometimes provide additional information about steps in a reaction pathway. In the case of bisubstratereactions, steady-state kinetics can help determine whether a ternarycomplex is formed during the reaction (Fig. 8-14). A more completepicture generally requires more sophisticated kinetic methods that gobeyond the scope of an introductory text. Here, we briefly introduce oneof the most important kinetic approaches for studying reaction mechanisms, pre-steady state kinetics.A complete description of an enzyme-catalyzed reaction requiresdirect measurement of the rates of individual reaction steps, for example the measurement of the association of enzyme and substrate toform the ES complex.
It is during the pre-steady state that the rates ofmany reaction steps can be measured independently. Reaction conditions are adjusted to facilitate the measurement of events that occurduring the reaction of a single substrate molecule. Because the presteady state phase of a reaction is generally very short, this often requires specialized techniques for very rapid mixing and sampling. Oneobjective is to gain a complete and quantitative picture of the energeticcourse of a reaction. As we have already noted, reaction rates and equilibria are related to the changes in free energy that occur during theISJ VmMIncreasing[S2]Figure 8-14 Steady-state kinetic analysis ofbisubstrate reactions.
In these double-reciprocalplots (see Box 8-1), the concentration of substrate 1is varied while the concentration of substrate 2 isheld constant. This is repeated for several values of[S2], generating several separate lines. The linesintersect if a ternary complex is formed in the reaction (a), but are parallel if the reaction goesthrough a ping-pong or double-displacement pathway (b).220Part II Structure and Catalysisreaction. Measuring the rate of individual reaction steps defines howenergy is used by a specific enzyme, which represents an importantcomponent of the overall reaction mechanism.
In a number of cases ithas proven possible to measure the rates of every individual step in amultistep enzymatic reaction. Some examples of the application of presteady state kinetics are included in the descriptions of specific enzymes later in this chapter.Enzymes Are Subject to Reversibleand Irreversible InhibitionElCompetitive inhibitionEI + SNoncompetitive inhibitionUncompetitive inhibitionFigure 8-15 Three types of reversible inhibition.Competitive inhibitors bind to the enzyme's activesite.
Noncompetitive inhibitors generally bind at aseparate site. Uncompetitive inhibitors also bind ata separate site, but they bind only to the ES complex. Ki is the equilibrium constant for inhibitorbinding.Enzymes catalyze virtually every process in the cell, and it should notbe surprising that enzyme inhibitors are among the most importantpharmaceutical agents known. For example, aspirin (acetylsalicylate)inhibits the enzyme that catalyzes the first step in the synthesis ofprostaglandins, compounds involved in many processes including somethat produce pain.
The study of enzyme inhibitors also has providedvaluable information about enzyme mechanisms and has helped definesome metabolic pathways. There are two broad classes of enzyme inhibitors: reversible and irreversible.One common type of reversible inhibition is called competitive(Fig.
8-15). A competitive inhibitor competes with the substrate forthe active site of an enzyme, but a reaction usually does not occur oncethe inhibitor (I) is bound. While the inhibitor occupies the active site itprevents binding by the substrate. Competitive inhibitors are oftencompounds that resemble the substrate and combine with the enzymeto form an El complex (Fig. 8-15). This type of inhibition can be analyzed quantitatively by steady-state kinetics (Box 8-2).
Because theinhibitor binds reversibly to the enzyme, the competition can be biasedto favor the substrate simply by adding more substrate. When enoughsubstrate is present the probability that an inhibitor molecule willbind is minimized, and the reaction exhibits a normal Vmax. However,the [S] at which Vo = iV max , the Km, will increase in the presence ofinhibitor. This effect on the apparent Km and the absence of an effecton y m a x is diagnostic of competitive inhibition, and is readily revealedin a double-reciprocal plot (Box 8-2). The equilibrium constant for inhibitor binding, Kh can be obtained from the same plot.Competitive inhibition is used therapeutically to treat patientswho have ingested methanol, a solvent found in gas-line antifreeze.Methanol is converted to formaldehyde by the action of the enzymealcohol dehydrogenase.
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