2 Структура и функция белка (1160071), страница 32
Текст из файла (страница 32)
This use of weak-bondinginteractions between the metal and the substrate is similar to some ofthe uses of enzyme-substrate binding energy described earlier. Metalscan also mediate oxidation-reduction reactions by reversible changesin the metal ion's oxidation state. Nearly a third of all known enzymesrequire one or more metal ions for catalytic activity.Chapter 8 EnzymesChymotrypsinr:ESer195HO 7Ser195BH(/RS-N^-C-R1IIIHOFigure 8-10 The first step in the reaction catachymotrypsin, also called the acylationstep. The hydroxyl group of Ser195 is the nucleophile in a reaction aided by general base catalysis(the base is the side chain of His57). The chymotrypsin reaction is described in more detail in Fig.8 19~ Jyzed b y,+2R -NH 2A combination of several catalytic strategies is usually employedby an enzyme to bring about a rate enhancement.
A good example ofthe use of both covalent catalysis and general acid-base catalysis occurs in chymotrypsin. The first step in the reaction catalyzed by chymotrypsin is the cleavage of a peptide bond. This is accompanied byformation of a covalent linkage between a Ser residue on the enzymeand part of the substrate; this reaction is enhanced by general basecatalysis by other groups on the enzyme (Fig. 8-10).
The chymotrypsinreaction is described in more detail later in this chapter.Enzyme Kinetics as an Approach toUnderstanding MechanismMultiple approaches are commonly used to study the mechanism ofaction of purified enzymes. A knowledge of the three-dimensionalstructure of a protein provides important information. The value ofstructural information is greatly enhanced by classical protein chemistry and modern methods of site-directed mutagenesis (changing theamino acid sequence of a protein in a defined way by genetic engineering; see Chapter 28) that permit enzymologists to examine the role ofindividual amino acids in structure and enzyme action.
However, therate of the catalyzed reaction can also reveal much about the enzyme.The study of reaction rates and how they change in response tochanges in experimental parameters is known as kinetics. This is theoldest approach to understanding enzyme mechanism, and one thatremains most important today. The following is a basic introduction tothe kinetics of enzyme-catalyzed reactions. The more advanced student may wish to consult the texts and articles cited at the end of thischapter.Substrate Concentration Affects the Rate ofEnzyme-Catalyzed ReactionsA discussion of kinetics must begin with some fundamental concepts.One of the key factors affecting the rate of a reaction catalyzed by apurified enzyme in vitro is the amount of substrate present, [S].
Butstudying the effects of substrate concentration is complicated by thefact that [S] changes during the course of a reaction as substrate isconverted to product. One simplifying approach in a kinetic experiment is to measure the initial rate (or initial velocity), designated Vo,when [S] is generally much greater than the concentration of enzyme.Then, if the time is sufficiently short following the start of a reaction,changes in [S] are negligible, and [S] can be regarded as a constant.211Part II Structure and Catalysis212The effect on Vo of varying [S] when the enzyme concentration isheld constant is shown in Figure 8-11. At relatively low concentrationsof substrate, Vo increases almost linearly with an increase in [S]. Athigher substrate concentrations, Vo increases by smaller and smalleramounts in response to increases in IS].
Finally, a point is reachedbeyond which there are only vanishingly small increases in Vo withincreasing [S] (Fig. 8-11). This plateau is called the maximum velocity,'max-KmSubstrateconcentration, [S] (mM)Figure 8-11 Effect of substrate concentration onthe initial velocity of an enzyme-catalyzed reaction.Vmax can only be approximated from such a plot,because Vo will approach but never quite reachVmax. The substrate concentration at which Vo ishalf maximal is Km, the Michaelis-Menten constant.
The concentration of enzyme E in an experiment such as this is generally so low that [S] : »[E] even when [S] is described as low or relativelylow. The units given are typical for enzyme-catalyzed reactions and are presented only to help illustrate the meaning of Vo and [S]. (Note that thecurve describes part of a rectangular hyperbola,with one asymptote at Vmax. If the curve were continued below [S] = 0, it would approach a verticalasymptote at [S] - —Km.)Leonor Michaelis1875-1949Maud Menten1879-1960The ES complex is the key to understanding this kinetic behavior,just as it represented a starting point for the discussion of catalysis.The kinetic pattern in Figure 8-11 led Victor Henri to propose in 1903that an enzyme combines with its substrate molecule to form the EScomplex as a necessary step in enzyme catalysis.
This idea was expanded into a general theory of enzyme action, particularly by LeonorMichaelis and Maud Menten in 1913. They postulated that the enzymefirst combines reversibly with its substrate to form an enzymesubstrate complex in a relatively fast reversible step:E + SES(8-7)The ES complex then breaks down in a slower second step to yield thefree enzyme and the reaction product P:ESk;E + P(8-8)In this model the second reaction (Eqn 8-8) is slower and thereforelimits the rate of the overall reaction. It follows that the overall rate ofthe enzyme-catalyzed reaction must be proportional to the concentration of the species that reacts in the second step, that is, ES.At any given instant in an enzyme-catalyzed reaction, the enzymeexists in two forms, the free or uncombined form E and the combinedform ES.
At low [S], most of the enzyme will be in the uncombined formE. Here, the rate will be proportional to [S] because the equilibrium ofEquation 8-7 will be pushed toward formation of more ES as [S] isincreased. The maximum initial rate of the catalyzed reaction (Vmax)isobserved when virtually all of the enzyme is present as the ES complexand the concentration of E is vanishingly small. Under these conditions, the enzyme is "saturated" with its substrate, so that furtherincreases in [S] have no effect on rate. This condition will exist when[S] is sufficiently high that essentially all the free enzyme will havebeen converted into the ES form. After the ES complex breaks down toyield the product P, the enzyme is free to catalyze another reaction.The saturation effect is a distinguishing characteristic of enzyme catalysts and is responsible for the plateau observed in Figure 8-11.When the enzyme is first mixed with a large excess of substrate,there is an initial period called the pre-steady state during which theconcentration of the ES complex builds up.
The pre-steady state isusually too short to be easily observed. The reaction quickly achieves asteady state in which [ES] (and the concentration of any other intermediates) remains approximately constant over time. The measuredVo generally reflects the steady state even though Vo is limited to earlytimes in the course of the reaction. Michaelis and Menten concernedthemselves with the steady-state rate, and this type of analysis is re-ferred to as steady-state kinetics.The Relationship between Substrate Concentration andEnzymatic Reaction Rate Can Be Expressed QuantitativelyFigure 8-11 shows the relationship between [S] and Vo for an enzymatic reaction. The curve expressing this relationship has the samegeneral shape for most enzymes (it approaches a rectangular hyperbola). The hyperbolic shape of this curve can be expressed algebraicallyby the Michaelis-Menten equation, derived by these workers startingfrom their basic hypothesis that the rate-limiting step in enzymaticreactions is the breakdown of the ES complex to form the product andthe free enzyme.The important terms are [SJ, Vo, Vmax, and a constant called theMichaelis-Menten constant or Km.
All of these terms are readily measured experimentally.Here we shall develop the basic logic and the algebraic steps in amodern derivation of the Michaelis-Menten equation. The derivationstarts with the two basic reactions involved in the formation andbreakdown of ES (Eqns 8-7 and 8-8). At early times in the reaction,the concentration of the product [P] is negligible and the simplifyingassumption is made that £- 2 can be ignored.
The overall reaction thenreduces toE+S .kl- ESkl> E+P(8-9)k ,Vo is determined by the breakdown of ES to give product, which isdetermined by [ES]:Vo = &2[ES](8-10)As [ESJ in Equation 8-10 is not easily measured experimentally, wemust begin by finding an alternative expression for [ES]. First, we willintroduce the term [EJ, representing the total enzyme concentration(the sum of the free and substrate-bound enzyme). Free or unboundenzyme can then be represented by [Et] - [ES].
Also, because [S] isordinarily far greater than [Et], the amount of substrate bound by theenzyme at any given time is negligible compared with the total [S].With these in mind, the following steps will lead us to an expression forVo in terms of parameters that are easily measured.Step 1.
The rates of formation and breakdown of ES are determinedby the steps governed by the rate constants k\ (formation) and k-i + k2(breakdown), according to the expressionsRate of ES formation = iWfEtl - IES|)[S1(8-11)Rate of ES breakdown = Jfe^tES] + &2[ES1(8-12)Step 2. An important assumption is now made that the initial rate ofreaction reflects a steady state in which [ES] is constant, i.e., the rateof formation of ES is equal to its rate of breakdown.
This is called thesteady-state assumption. The expressions in Equations 8-11 and 8-12can be equated at the steady state, givingkx([Et] - [ESJ)[S] = fc-ilES] + &2[ES](8-13)Step 3. A series of algebraic steps is now taken to solve Equation 8-13for [ES]. The left side is multiplied out and the right side is simplifiedto give(8-14)214Part II Structure and CatalysisAdding the terming givesto both sides of the equation and simplifyWEtlLS] = (&JS] + k-x + &2)[ES](8-15)Solving this equation for [ES] gives(8-16)This can now be simplified further, in such a way as to combine therate constants into one expression:[EJ[S][S] + (k2 + k-1)/k1[ES] =(8-17)The term (k2 + k-i)lki is defined as the Michaelis-Menten constant, Km.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
