2 Структура и функция белка (1160071), страница 37
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When a transition-state analog isin the transition state. These studies provide addiused as an antigen to stimulate the production oftional evidence for enzyme-transition state comantibodies, the antibodies that bind it are potentialplementarity and suggest that new classes of anticatalysts of the corresponding reaction. This apbody catalysts might be developed for research andproach, first suggested by William P.
Jencks inindustry.1969, has become practical with the development ofSubstrate (a polypeptidt"226• : - . • ( •CM X H A AProduct 2t()('CHAsp : '--C'XH AA(a)"*XxH()-Ser!'"'AA,C CH -XHC-CH-NH-AA,,H H \^JXX'Oxyanion holeHvdrophobi. pocketGly""W ^HO-C--CH-NH--AA,,56N ^ >;xxG\\""Ser1""'iiChymotrypsin 'free enzyme'Enzyme—substrate complexHXEnzyme-product 2 complex'XH.A A,(: • i "H\UO K() -Sor'-'C-CH-NH-AA*6* VTransition stateH-O-C—CH-- NH-AA,,lacvlation >Asp:"-' - CProduct 1H-O'»C-CH-NH--AA.,HXC-CH-NH-AA*HHXXH^XTransition state 'deacylation'Acyl-enzyme intermediateFigure 8-19 Steps in the cleavage of a peptidebond by chymotrypsin.
The substrate (a polypeptideor protein) is bound at the active site. The peptidebond to be cleaved is positioned by the binding ofthe adjacent hydrophobic amino acid side chain (aPhe residue in this example) in a special hydrophobic pocket on the enzyme, as shown. The reactionconsists of two phases: (a) to (c) formation of acovalent acyl-enzyme intermediate coupled to cleavage of the peptide bond (the acylation phase) and(d) to (g), deacylation to regenerate the free enzyme (the deacylation phase). In both phases, thecarbonyl oxygen of the substrate acquires a negative charge in the transition state. The charge isstabilized by a hydrogen bond to the amide nitrogens of Gly193 and Ser195; the hydrogen bond toGly193 forms only in the transition state. Deacylation is essentially the reverse of acylation, withwater serving in place of the amine component ofthe substrate.
The His and Asp residues cooperatein a catalytic triad, providing general base catalysisof steps (b) and (e) and general acid catalysis ofsteps (c) and (f).Acyl-enzyme intermediateThe enzyme reaction of chymotrypsin has two major phases: acylation,in which the peptide bond is cleaved and an ester linkage is formedbetween the peptide carbonyl carbon and the enzyme; and deacylation,in which the ester linkage is hydrolyzed and the enzyme regenerated.The nucleophile in the acylation phase is the oxygen of Ser195. A serinehydroxyl is normally protonated at neutral pH, but in the enzymeSer195 is hydrogen-bonded to His 57 , which is further hydrogen-bondedto Asp102.
These three amino acids are often referred to as a catalytictriad. As the serine oxygen attacks the carbonyl carbon of a peptidebond, the hydrogen-bonded His 57 functions as a general base to abstract the serine proton, and the negatively charged Asp102 stabilizesthe positive charge that forms on the His residue. This prevents thedevelopment of a very unstable positive charge on the serine hydroxyland increases its nucleophilicity. His 57 can also act as a proton donor toprotonate the amino group in the displaced portion of the substrate(the leaving group). A similar set of proton transfers occurs in the deacylation step (Fig. 8-19).As the serine oxygen attacks the carbonyl group in the substrate, atransition state is reached in which the carbonyl oxygen acquires anegative charge. This charge is formed within a pocket on the enzymecalled the oxyanion hole, and it is stabilized by hydrogen bonds contributed by the amide nitrogens of two peptide bonds in the proteinbackbone.
One of these hydrogen bonds occurs only in the transitionstate and thereby reduces the energy required to reach the transitionstate. This represents an example of the use of binding energy in catalysis. The importance of binding energy in catalysis by chymotrypsin isdiscussed further in Box 8-3.Chapter 8 EnzymesThe first evidence for a covalent acyl-enzyme intermediate camefrom a classic application of pre-steady state kinetics. In addition to itsaction on polypeptides, chymotrypsin will catalyze the hydrolysis ofsmall ester and amide compounds.
These reactions are much slowerbecause less binding energy is available with these substrates, butthey are easier to study. Studies by B.S. Hartley and B.A. Kilby foundthat the hydrolysis of p-nitrophenylacetate by chymotrypsin, as measured by release of p-nitrophenol, proceeded with a rapid burst beforeleveling off to a slower rate (Fig.
8-20). By extrapolating back to zerotime, they concluded that the burst phase corresponded to just underone molecule of p-nitrophenol released for every enzyme molecule present. They suggested that this reflected a rapid acylation of all the enzyme molecules (with release of p-nitrophenol) but that subsequentturnover of the enzyme was limited in rate by a slow deacylation step.Similar results have been obtained with many enzymes.Hexokinase This is a bisubstrate enzyme (Mr 100,000), catalyzing theinterconversion of glucose and ATP with glucose-6-phosphate andADP. The hydroxyl at position 6 of the glucose molecule (to which they-phosphate of ATP is transferred) is similar in chemical reactivity towater, and water freely enters the enzyme active site. Yet hexokinasediscriminates between glucose and water, with glucose favored by afactor of 106.HMg•ATP +HCH 2 OH-OHOH HHHCH 2 OPO|-O-^ Mg • ADP +2273.0^2.0oI1.0zgi12Time (min)O9NCH 3 —C-OHAcetic acidOHHOHGlucoseHOHGlucose-6-phosphateHexokinase can discriminate between glucose and water becauseof a conformational change in the enzyme that occurs when the correctsubstrates are bound (Fig.
8-21). The enzyme thus provides a goodexample of induced fit. When glucose is not present, the enzyme is inan inactive conformation with the active-site amino acid side chainsout of position for reaction. When glucose (but not water) and ATPbind, the binding energy derived from this interaction induces achange to the catalytically active enzyme conformation.TyrosyltRNA Synthetase This enzyme (Mr 95,000) catalyzes the attachment of tyrosine to an RNA molecule called a transfer RNA, activating the amino acid to form a precursor for protein synthesis (described in Chapter 26).
The reaction proceeds in two phases:Enz + Tyr + ATP ^Enz • Tyr-AMP + tRNAFigure 8-20 Observed kinetics of the hydrolysisof p-nitrophenylacetate (p-NPA) by chymotrypsinas measured by release of p-nitrophenol (a coloredproduct). A rapid release (burst) of an amount ofp-nitrophenol nearly stoichiometric with theamount of enzyme present is observed. This reflectsthe fast acylation phase of the reaction. The subsequent rate is slower because enzyme turnover islimited by the rate of the slower deacylation phase.3&*TEnz • Tyr-AMP + PP,± Tyr-tRNA + Enz + AMP(PPi is the abbreviation for inorganic pyrophosphate. Pi? used later inthis chapter, is the abbreviation for inorganic phosphate.) Kineticstudies have shown that ATP and tyrosine bind to the enzyme in random order. The tyrosyl-AMP intermediate is not released by the enzyme, and is sufficiently stable to allow study of the first reactionphase in isolation. The following discussion focuses on this phase.Figure 8—21 The conformational change induced inhexokinase by the binding of a substrate (D-glucose,shown in blue).Figure 8-22 The structure of tyrosyl-tRNA synthetase.
At left, the enzyme is shown without substrate bound. Active site residues that hydrogenbond to the tyrosyl-AMP intermediate (Fig. 8-23a)are shown in red, and two residues (Thr40 andHis45) that contribute hydrogen bonds in the reaction transition state (Fig. 8-23b) are shown in orange. The amino acid side chains of Lys82 andArg86, which cover part of the active site, are notshown in order to expose more of the active siteresidues. At right, the same view is shown with thetyrosyl—AMP intermediate (shown in blue, with aphosphorus atom in yellow) bound.The structure of tyrosyl-tRNA synthetase in its complex withtyrosyl-AMP is shown in Figure 8-22. This structure indicates a number of potential hydrogen bonds between enzyme and substrate (Fig.8-23).
Alan Fersht and colleagues have used this information to create, by site-directed mutagenesis (Chapter 28), a series of mutant enzymes lacking one or another of the amino acid side chains contributing to these hydrogen bonds.Hydrogen bonds formed in the ES complex affect Ks, which can bedirectly measured for this enzyme. Hydrogen bonds formed only in thetransition state affect kcat.
In several cases, substitution of a nonhydrogen-bonding amino acid at key positions in the active site affectsHisGln17:r— of%QO_'Asp' H /-C—O00\ + H ||||H—N— C—C—O—P—O—CHHTyr^-OHTyrosyl-AMPHO—; Thr11Cys:OAsp17"}—C—OTyrosyl-tRNA synthetase(a)OHH.N-C-C-O"vThr40OH OHEnzyme-tyrosine-ATPcomplexHO-iThr 4HNOHFigure 8-23 (a) Hydrogen bonding between sidechains of tyrosyl-tRNA synthetase and tyrosyl-AMP,as deduced from x-ray crystallographic studies,(b) Hydrogen bonds between enzyme and substratethat stabilize the transition state in the reactionleading to formation of tyrosyl-AMP on tyrosyltRNA synthetase.
Amino acids that contribute hydrogen bonds primarily in the transition state areshown in red.OH OHTransition state+ HH,N-C-C-O-P;(b)OHOH OHEnzyme-tyrosyl-AMP complexHis45Chapter 8 Enzymes229kcat but does not affect Ks. For example, substitution of an alanine forThr40 and a glycine for His 45 has little effect on the observed Ks forATP. However, these substitutions lower the kcat for the reaction by afactor of 300,000. In other words, this altered enzyme can still bind itssubstrates to form the ES complex, but once bound, the substrates donot react as rapidly because the enzyme can no longer form two essential hydrogen bonds that normally help to lower the activation energyrequired to reach the transition state (Fig.
8-23).Regulatory EnzymesWe now turn to a special class of enzymes that represent exceptions tosome of the rules outlined so far in this chapter. In cell metabolism,groups of enzymes work together in sequential pathways to carry out agiven metabolic process, such as the multireaction conversion of glucose into lactate in skeletal muscle or the multireaction synthesis of anamino acid from simpler precursors in a bacterial cell. In such enzymesystems, the reaction product of the first enzyme becomes the substrate of the next, and so on (Figure 8-24).Most of the enzymes in each system follow kinetic patterns alreadydescribed.
In each enzyme system, however, there is at least one enzyme that sets the rate of the overall sequence because it catalyzes theslowest or rate-limiting reaction. These regulatory enzymes exhibitincreased or decreased catalytic activity in response to certain signals.By the action of such regulatory enzymes, the rate of each metabolicsequence is constantly adjusted to meet changes in the cell's demandsfor energy and for biomolecules required in cell growth and repair. Inmost multienzyme systems the first enzyme of the sequence is a regulatory enzyme.
Catalyzing even the first few reactions of a pathwaythat leads to an unneeded product diverts energy and metabolites frommore important processes. An excellent place to regulate a metabolicpathway, therefore, is at the point of commitment to the pathway. Theother enzymes in the sequence are usually present in amounts providing a large excess of catalytic activity; they can promote their reactionsonly as fast as their substrates are made available from precedingreactions.The activity of regulatory enzymes is modulated through varioustypes of signal molecules, which are generally small metabolites orcofactors.
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