2 Структура и функция белка (1160071), страница 36

Просмтор этого файла доступен только зарегистрированным пользователям. Но у нас супер быстрая регистрация: достаточно только электронной почты!

Текст из файла (страница 36)

The discussion concentrates on selected principles, along with some key experiments thathave helped to bring them into focus. Much mechanistic detail andexperimental evidence is omitted, and in no instance do the mechanisms described below provide a complete explanation for the catalyticrate enhancements brought about by these enzymes.220245(a)Chymotrypsin This enzyme is a protease (Mr 25,000) specific for peptide bonds adjacent to aromatic amino acid residues (see Table 6-7).The three-dimensional structure of chymotrypsin is shown in Figure8-18, with functional groups in the active site emphasized. This enzyme reaction illustrates the principle of transition-state stabilizationby an enzyme, and also provides a classic example of the use of generalacid-base catalysis and covalent catalysis (Fig.

8-19, p. 226).Figure 8—18 The structure of chymotrypsin. (a) Arepresentation of primary structure, showing disulfide bonds and the location of key amino acids.Note that the protein consists of three polypeptidechains. The active-site amino acids are foundgrouped together in the three-dimensional structure, (b) A space-filling model of chymotrypsin. Thepocket in which the aromatic amino acid side chainis bound is shown in green.

The amide nitrogens ofGly193 and Ser195 in the polypeptide backbone makeup the oxyanion hole (see Fig. 8-19), and areshown in orange. The side chains of other key active site residues, including Ser195, His57, andAsp102 are shown in red, and are explained in Fig.8-19. (c) The polypeptide backbone of chymotrypsinshown as a ribbon structure. Disulflde bonds areshown in yellow; the A, B, and C chains are shownin dark blue, light blue, and white, respectively.(d) A close-up of the chymotrypsin active site witha substrate bound.

Ser195 attacks the carbonylgroup of the substrate (shown in purple); the developing negative charge on the oxygen is stabilizedby the oxyanion hole (amide nitrogens shown inorange), as explained in Fig. 8—19. In the substrate,the aromatic amino acid side chain and the amidenitrogen of the peptide bond to be cleaved areshown in light blue.224BOXPart II Structure and Catalysis8-3Evidence for Enzyme-Transition State ComplementarityThe transition state of a reaction is difficult tostudy, because by definition it has no finite lifetime. To understand enzymatic catalysis, however,we must dissect the interaction between the enzyme and this ephemeral moment in the course ofa reaction.

The idea that an enzyme is complementary to the transition state is virtually a requirement for catalysis, because the energy hill uponwhich the transition state sits is what the enzymemust lower if catalysis is to occur. How can we obtain evidence that the idea of enzyme-transitionstate complementarity is really correct? Fortunately, there are a variety of approaches, old andnew, to this problem. Each has provided compelling evidence in support of this general principle ofenzyme action.Structure-Activity CorrelationsFigure 1 Effects ofsmall structuralchanges in the substrate on kinetic parameters for chymotrypsin-catalyzed amidehydrolysis.motrypsin normally catalyzes the hydrolysis ofpeptide bonds next to aromatic amino acids, andthe substrates shown in Figure 1 are convenientsmaller models for the natural substrates (longpolypeptides and proteins; see Chapter 6).

Theadditional chemical groups added in going from Ato B to C are shaded in red. Note that the interaction between the enzyme and these added functional groups has a minimal effect on Km (which istaken here as a reflection of Ks), but a large, positive effect on kcat and kcat/Km. This is what wewould expect if the interaction occurred only inthe transition state. Chymotrypsin is described inmore detail beginning on page 223.A complementary experimental approach tothis problem is to modify the enzyme, eliminatingcertain enzyme-substrate interactions, by replacing specific amino acids through site-directed mutagenesis (Chapters 7 and 28).

A good example isfound in tyrosyl-tRNA synthetase (p. 227).If enzymes are complementary to reaction transition states, then some functional groups in the substrate and in the enzyme must interact preferentially with the transition state rather than the EScomplex. Altering these groups should have littleeffect on formation of the ES complex, and henceshould not affect kinetic parameters (K$, or sometimes Km ifKs = Km) that reflect the E + S ^ ESequilibrium. Changing the same groups, however,should have a large effect on the overall rate (kcator kcat/Km) of the reaction, because the bound substrate lacks potential binding interactions neededto lower the activation energy.An excellent example is seen in a series of substrates for the enzyme chymotrypsin (Fig.

1). Chy-Even though transition states cannot be observeddirectly, chemists can often predict the approximate structure of a transition state based on accumulated knowledge about reaction mechanisms.The transition state by definition is transient andso unstable that direct measurement of the bindinginteraction between this species and the enzyme isimpossible. In some cases, however, stable molecules can be designed that resemble transitionstates.

These are called transition-state analogs. In principle, they should bind to an enzymeO(s" 1 )(mM)CH3 —C—NH—CH—C—NH20.0631OOCH 2 OIIIIIIICH3 —C-NH-CH-C—NH—CH 2 —C—NH 20.1415102.825114Substrate ASubstrate BoSubstrate CTransition-State AnalogsCH2 OCH2 OOvT1 s" 1 )CH3 OCH 3 —C—NH—CH—C—NH—CH—C—NH2Chapter 8 Enzymes225OHAEster hydrolysisIIII0>~R2> Products"R26STransition stateP\2AnalogOHProductsCarbonate hydrolysisFigure 2 The expectedtransition states forester or carbonate hydrolysis reactions.Phosphonate and phosphate compounds, respectively, make goodtransition-state analogsfor these reactions.Transition stateHH-N'.0 ii O/HNO.Analogmore tightly than the substrate binds in the EScomplex, because they should fit in the active sitebetter (i.e., form more weak interactions) than thesubstrate itself.

The idea of transition-state analogs was suggested by Pauling in the 1940s, and ithas been used for a number of enzymes. These experiments have the limitation that a transitionstate analog can never mimic a transition stateperfectly. Analogs have been found, however, thatbind an enzyme 102 to 106 times more tightly thanthe normal substrate, providing good evidence thatenzyme active sites are indeed complementary totransition states.laboratory techniques to produce antibodies thatare all identical and bind one specific antigen(these are known as monoclonal antibodies; seeChapter 6).Pioneering work in the laboratories of RichardLerner and Peter Schultz has resulted in the isolation of a number of monoclonal antibodies that catalyze the hydrolysis of esters or carbonates (Fig.

2).In these reactions, the attack by water (0H~) onthe carbonyl carbon produces a tetrahedral transition state in which a partial negative charge hasdeveloped on the carbonyl oxygen. Phosphonatecompounds mimic the structure and charge distribution of this transition state in ester hydrolysis,making them good transition-state analogs; phosCatalytic Antibodiesphate compounds are used for carbonate reactions.If a transition-state analog can be designed for theAntibodies that bind the phosphonate or phosreaction S —> P, then an antibody that binds tightly phate tightly have been found to catalyze the corto the transition-state analog might catalyzeresponding ester or carbonate hydrolysis reactionS —> P.

Antibodies (immunoglobulins; see Fig. 6-8) by factors of 103 to 104. Structural analyses of a feware key components of the immune response. Aof these catalytic antibodies have shown that themolecule or chemical group that is bound tightlycatalytic amino acid side chains are arrangedand specifically by a given antibody is referred towhere they could interact with the substrate onlyas an antigen.

Характеристики

Список файлов лекций