2 Структура и функция белка (1160071), страница 31
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Theenzyme must provide functional groups for ionic interactions, hydrogen bonds, and other interactions, and also precisely position thesegroups so that binding energy is optimized in the transition state.Enzymes Use Binding Energy to ProvideReaction Specificity and CatalysisCan binding energy account for the huge rate accelerations broughtabout by enzymes? Yes.
As a point of reference, Equation 8-6 allows usto calculate that about 5.7 kJ/mol of free energy is required to accelerate a first-order reaction by a factor of ten under conditions commonlyfound in cells. The energy available from formation of a single weakinteraction is generally estimated to be 4 to 30 kJ/mol. The overallenergy available from formation of a number of such interactions canlower activation energies by the 60 to 80 kJ/mol required to explain thelarge rate enhancements observed for many enzymes.The same binding energy that provides energy for catalysis alsomakes the enzyme specific. Specificity refers to the ability of an enzyme to discriminate between two competing substrates.
Conceptually,this idea is easy to distinguish from the idea of catalysis. Catalysis andspecificity are much more difficult to distinguish experimentally because they arise from the same phenomenon. If an enzyme active sitehas functional groups arranged optimally to form a variety of weakinteractions with a given substrate in the transition state, the enzymewill not be able to interact as well with any other substrate. For example, if the normal substrate has a hydroxyl group that forms a specifichydrogen bond with a Glu residue on the enzyme, any molecule lackingthat particular hydroxyl group will generally be a poorer substrate forthe enzyme.
In addition, any molecule with an extra functional groupfor which the enzyme has no pocket or binding site is likely to be excluded from the enzyme. In general, specificity is also derived from theformation of multiple weak interactions between the enzyme andmany or all parts of its specific substrate molecule.(t)AG^/sX \AGBuncatcat- ES E P " \PReaction coordinateFigure 8—7 The role of binding energy in catalysis.To lower the activation energy for a reaction, thesystem must acquire an amount of energy equivalent to the amount by which AG^ is lowered.
Thisenergy comes largely from binding energy (AGB)contributed by formation of weak noncovalent interactions between substrate and enzyme in the transition state. The role of AGB is analogous to that ofAGM in Fig. 8-6.CatalysisThe general principles outlined above can be illustrated by a variety of recognized catalytic mechanisms. These mechanisms are notmutually exclusive, and a given enzyme will often incorporate severalin its own complete mechanism of action. It is often difficult to quantifythe contribution of any one catalytic mechanism to the rate and/orspecificity of an enzyme-catalyzed reaction.Binding energy is the dominant driving force in several mechanisms, and these can be the major, and sometimes the only, contribution to catalysis.
This can be illustrated by considering what needs tooccur for a reaction to take place. Prominent physical and thermodynamic barriers to reaction include (1) entropy, the relative motion oftwo molecules in solution; (2) the solvated shell of hydrogen-bondedwater that surrounds and helps to stabilize most biomolecules in aqueous solution; (3) the electronic or structural distortion of substratesthat must occur in many reactions; and (4) the need to achieve properalignment of appropriate catalytic functional groups on the enzyme.Binding energy can be used to overcome all of these barriers.A large reduction in the relative motions of two substrates that areto react, or entropy reduction, is one of the obvious benefits of binding them to an enzyme.
Binding energy holds the substrates in theproper orientation to react—a major contribution to catalysis becauseproductive collisions between molecules in solution can be exceedinglyrare. Substrates can be precisely aligned on the enzyme. A multitude ofweak interactions between each substrate and strategically locatedgroups on the enzyme clamp the substrate molecules into the properpositions. Studies have shown that constraining the motion of two reactants can produce rate enhancements of as much as 108 M (a rateequivalent to that expected if the reactants were present at the impossibly high concentration of 100,000,000 M).Formation of weak bonds between substrate and enzyme also results in desolvation of the substrate. Enzyme-substrate interactionsreplace most or all of the hydrogen bonds that may exist between thesubstrate and water in solution.Binding energy involving weak interactions formed only in the reaction transition state helps to compensate thermodynamically for anystrain or distortion that the substrate must undergo to react.
Distortion of the substrate in the transition state may be electrostatic orstructural.The enzyme itself may undergo a change in conformation when thesubstrate binds, induced again by multiple weak interactions with thesubstrate. This is referred to as induced fit, a mechanism postulatedby Daniel Koshland in 1958. Induced fit may serve to bring specificfunctional groups on the enzyme into the proper orientation to catalyzethe reaction.
The conformational change may also permit formation ofadditional weak-bonding interactions in the transition state. In eithercase the new conformation may have enhanced catalytic properties.Specific Catalytic Groups Contribute to CatalysisOnce a substrate is bound, additional modes of catalysis can be employed by an enzyme to aid bond cleavage and formation, using properly positioned catalytic functional groups. Among the best characterized mechanisms are general acid-base catalysis and covalentcatalysis. These are distinct from mechanisms based on binding energy because they generally involve covalent interaction with a substrate, or group transfer to or from a substrate.Chapter 8 EnzymesGeneral Acid-Base Catalysis Many biochemical reactions involvethe formation of unstable charged intermediates that tend to breakdown rapidly to their constituent reactant species, thus failing to undergo reaction (Fig.
8-8). Charged intermediates can often be stabilized (and the reaction thereby catalyzed) by transferring protons to orfrom the substrate or intermediate to form a species that breaks downto products more readily than to reactants. The proton transfers caninvolve the constituents of water alone or may involve other weak proton donors or acceptors. Catalysis that simply involves the H + (H3O + )or 0H~ ions present in water is referred to as specific acid or basecatalysis. If protons are transferred between the intermediate andwater faster than the intermediate breaks down to reactants, the intermediate will effectively be stabilized every time it forms.
No additional catalysis mediated by other proton acceptors or donors willoccur. In many cases, however, water is not enough. The term generalacid-base catalysis refers to proton transfers mediated by otherclasses of molecules. It is observed in aqueous solutions only when theunstable reaction intermediate breaks down to reactants faster thanR1R3H - C - O H + C=Oif"- "N—HA-R1R3I H |H—C—O—C—O~R2N—Hspecific acid-basecatalysisR1Without catalysis,unstable (charged)intermediate breaksdown rapidlyto form reactants.BHA_When proton transferto or from H2O is slowerthan the rate of breakdownof intermediates, only a13RRBH fraction of the intermediates formed will beIIstabilized. The presence ofH—C-O-C-0alternative proton donorsR2 H - N ^ Hor acceptors increasesthe rate of the reaction.R3 HOHH-C-O-C-O"R2 H-N^HA4Figure 8-8 Unfavorable charge development during cleavage of an amide.
This type of reaction iscatalyzed by chymotrypsin and other proteases.Charge development can be circumvented by donation of a proton by H3O* (specific acid catalysis) orby HA (general acid catalysis), where HA represents any acid. Similarly, charge can be neutralizedby proton abstraction by OH" (specific base catalysis) or by B : (general base catalysis), where B :represents any base.general acid-basecatalysisOH'pftien proton transferjgp or from H2O isBister than thefcate of breakdownppf intermediates,p||ie presence ofipher proton donors&r acceptors does notIncrease the rate ofpie reaction.Reactants eciesPH2OR1R3IIH—C—O-C=OH\NIH/Products209210Figure 8-9 Many organic reactions are promotedby proton donors (general acids) or proton acceptors(general bases). The active sites of some enzymescontain amino acid functional groups, such as thoseshown here, that can participate in the catalyticprocess as proton donors or proton acceptors.Glu, AspR-COOHR-COO"Lys, ArgHR-NHHR-NH 2CysR-SHR-S"R-C=CHR-C=CHHisHN^^NtHHTyrAmino acidresiduesGeneral acid form(proton donor)General base form(proton acceptor)the rate of proton transfer to or from water.
A variety of weak organicacids can supplement water as proton donors in this situation, or weakorganic bases can serve as proton acceptors. A number of amino acidside chains can similarly act as proton donors and acceptors (Fig. 8-9).These groups can be precisely positioned in an enzyme active site toallow proton transfers, providing rate enhancements on the order of102 to 105.Covalent Catalysis This involves the formation of a transient covalent bond between the enzyme and substrate.
Consider the hydrolysisof a bond between groups A and B:A—BH,0A+BIn the presence of a covalent catalyst (an enzyme with a nucleophilicgroup X:) the reaction becomesA—B + X:A—X + BA + X: + BThis alters the pathway of the reaction and results in catalysis onlywhen the new pathway has a lower activation energy than the uncatalyzed pathway. Both of the new steps must be faster than the uncatalyzed reaction. A number of amino acid side chains (including all ofthose in Fig.
8-9), as well as the functional groups of some enzymecofactors, serve as nucleophiles on some enzymes in the formation ofcovalent bonds with substrates. These covalent complexes always undergo further reaction to regenerate the free enzyme. The covalentbond formed between the enzyme and the substrate can activate asubstrate for further reaction in a manner that is usually specific tothe group or coenzyme involved.
The chemical contribution to catalysisprovided by individual coenzymes is described in detail as each coenzyme is encountered in Part III of this book.Metal Ion Catalysis Metals, whether tightly bound to the enzyme ortaken up from solution along with the substrate, can participate incatalysis in several ways. Ionic interactions between an enzyme-boundmetal and the substrate can help orient a substrate for reaction orstabilize charged reaction transition states.
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