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+ [S]cocAs IS] is increasedto higher and higher levels,essentiallyall ofthe enzymewill be bound to substrateat steadystate;at thispoint, a maximumrate of reaction,V-"r, will be reachedwhereV = V^u, = k."1[E6J.Thus,it is convenientto rewrite theMichaelis-Mentenequationastime +pre-steadystate:E Sf o r m i n gsteadystate:ESalmostconstant164Chapter3: Proteins\ir,HoSLOWoFAsr lltlc.\HoHnHc-,,/-,/,\H-NC--NHOHHO,/\HollFAsr\/FRVFAST\H/ \HHHCcHHOH-N(B)acid catalysis(C)II,,/.- \H/\-o//base catalysisilHHH\//oCI(A) no catalysisO(I(D)both acid andbase catalysesthat can be easily isolated in large quantities.
For these reasons, it has beenintensively studied, and it was the first enzyme to have its structure worked outin atomic detail by x-ray crystallography.The reaction that lysozyme catalyzes is a hydrolysis: it adds a molecule ofwater to a single bond between two adjacent sugar groups in the polysaccharidechain, thereby causing the bond to break (seeFigure 2-19). The reaction is energetically favorable because the free energy of the severedpolysaccharide chainis lower than the free energy of the intact chain.
However, the pure polysaccharide can remain for years in water without being hydrolyzed to any detectabledegree.This is because there is an energy barrier to the reaction, as discussedinChapter 2 (seeFigure 2-46). Acolliding water molecule can break a bond linkingtvvo sugars only if the polysaccharide molecule is distorted into a particularshape-the transition state-in which the atoms around the bond have analtered geometry and electron distribution. Becauseof this distortion, randomcollisions must supply a very large activation energy for the reaction to takeplace.
In an aqueous solution at room temperature, the energy of collisionsalmost never exceeds the activation energy. Consequently, hydrolysis occursextremely slowly, if at all.This situation changes drastically when the polysaccharide binds tolysozyme.The active site of lysozyme,becauseits substrate is a polymer, is a longgroove that holds six linked sugars at the same time. As soon as the polysaccharide binds to form an enzyme-substrate complex, the enzyme cuts the polysaccharide by adding a water molecule across one of its sugar-sugar bonds.
Theproduct chains are then quickly released,freeing the enzyme for further cyclesof reaction (Figure 3-50).The chemistry of the binding of lysozl.rneto its substrate is the same as thatfor antibody binding to its antigen-the formation of multiple noncovalentFigure3-50 The reactioncatalyzedby lysozyme.(A)The enzymelysozyme(E)catalyzesthe cuttingof a polysaccharidechain,which is itssubstrate(S).Theenzymefirstbindsto the chainto form anenzyme-substratecomplex(ES)and then catalyzesthe cleavageof aspecificcovalentbond in the backboneof the polysaccharide,forminganenzyme-productcomplex(EP)that rapidlydissociates.Releaseof theseveredchain(the productsP)leavesthe enzymefreeto act on anothersubstratemolecule.(B)A space-fillingmodelof the lysozymemoleculeboundto a shortlengthof polysaccharidechainbeforecleavage.(B,courtesyof RichardJ.
Feldmann.)(A))+EE)*E+PFigure3-49 Acid catalysisand basecatalysis.(A)The start of the uncatalyzedreactionshownin Figure3-474,withb/ueindicatingelectrondistributioninthe waterand carbonylbonds.(B)An acidlikesto donatea proton(H+)to otheratoms.By pairingwith the carbonyloxygen,an acidcauseselectronsto moveawayfrom the carbonylcarbon,makingthis atom much moreattractiveto theelectronegativeoxygenof an attackingwatermolecule.(C)A baselikesto takeup H+.By pairingwith a hydrogenof theattackingwatermolecule,a basecauseselectronsto move toward the wateroxygen/makingit a betterattackinggroupfor the carbonylcarbon.(D)Bypositionedatomshavingappropriatelyon its surface,an enzymecan performboth acidcatalysisand basecatalysisatthe sametime.165PROTEINFUNCTIONbonds.
However,lysozyme holds its polysaccharide substrate in a particular way,so that it distorts one of the two sugarsin the bond to be broken from its normal,most stable conformation. The bond to be broken is also held close to twoamino acids with acidic side chains (a glutamic acid and an aspartic acid) withinthe active site.Conditions are thereby created in the microenvironment of the lysozymeactive site that greatly reduce the activation energy necessaryfor the hydrolysisto take place.
Figure 3-51 shows three central steps in this enzymatically catalyzed reaction.The enzyme stressesits bound substrate, so that the shape of one sugarmore closely resembles the shape of high-energy transition states formedduring the reaction.2. The negatively charged aspartic acid reactswith the Cl carbon atom on thedistorted sugar,and the glutamic acid donates its proton to the oxygen thatlinks this sugar to its neighbor. This breaks the sugar-sugar bond andleaves the aspartic acid side chain covalently linked to the site of bondcleavage.3. Aided by the negatively charged glutamic acid, a water molecule reactswith the Cl carbon atom, displacing the aspartic acid side chain and completing the process of hydrolysis.t.Figure3-51 Eventsat the active site oflysozyme.<TGGT>The top left and toprightdrawingsshowthe freesubstrateand the freeproducts,respectively,whereasthe otherthreedrawingsshoweventsat the enzymethe sequentialactivesite.Notethe changein theof sugarD in theconformationcomplex;this shapeenzyme-substratethe oxocarbeniumchangestabilizesion-liketransitionstatesrequiredforof the covalentformationand hydrolysisintermediateshownin the middlepanel.It is alsooossiblethat a carboniumionformsin step2, astheintermediateshownin thecovalentintermediatemiddlepanelhasbeendetectedonly witha syntheticsubstrate.(SeeD.J.Vocadloet2001.)al.,Nature412:835-838,The overall chemical reaction, from the initial binding of the polysaccharideon the surface of the enzyme through the final release of the severed chains,occurs many millions of times faster than it would in the absence of enzyme.Other enzymes use similar mechanisms to lower activation energies andspeed up the reactions they catalyze.In reactions involving two or more reactants, the active site also acts like a template, or mold, that brings the substratestogether in the proper orientation for a reaction to occur between them (FigurePRODUCTSSUBSTRATET h i ss u b s t r a t ei s a n o l i g o s a c c h a r i doef s i xs u g a r s ,l a b e l e dA - F .O n l y s u g a r sD a n d E a r e s h o w n i n d e t a i lT h e f i n a l p r o d u c t sa r e a n o l i g o s a c c h a r i doef f o u r s u g a r s(/eft) and a disaccharide(dght), produced by hydrolysis.cHzoHA B CrOotFcH2oH-o---\i_'IH,3oV\ (.I n t h e e n z y m e - s u b s t r a tceo m p l e x( E 5 ) t, h ee n z y m ef o r c e ss u g a rD i n t o a s t r a i n e dc o n f o r m a t i o nw, i t h G l u 3 5 p o s i t i o n e dt o s e r v ea sa n a c i dt h a t a t t a c k st h e a d j a c e n ts u g a r - s u g a rb o n d b y d o n a t i n ga p r o t o n ( H + )t o s u g a rE ,a n dA s o 5 2 o o i s e dt o a t t a c kt h e C 1 c a r b o na t o mT h e A s p 5 2 h a sf o r m e d a c o v a l e n tb o n d b e t w e e nt h e e n z y m ea n d t h e C 1c a r b o na t o m o f s u g a rDT h e G l u 3 5 t h e n p o l a r i z e sa w a t e r m o l e c u l e( r e d ) ,s o t h a t i t s o x y g e nc a n r e a d i l ya t t a c kt h e C 1c a r b o na t o m a n d d i s p l a c eA s o 5 2T h e r e a c t i o no f t h e w a t e r m o l e c u l e( r e d )c o m p l e t e st h e h y d r o l y s ias n d r e t u r n st h e e n z y m et o i t s i n i t i a ls t a t e ,f o r m i n gt h e f i n a l e n z y m e o r o d u c tc o m p l e x( E P ) .166Chapter3: ProteinsFigure3-52 Somegeneralstrategiesofenzyme catalysis.(A)Holding substratestogetherin a precisealignment.(B)Chargestabilizationof reaction(C)Applyingforcesthatintermediates.distortbondsin the substrateto increasethe rateof a particularreaction.( A ) e n z y m eb i n d st o t w os u b s t r a t em o l e c u l e sa n do r i e n t st h e m p r e c i s e ltyoe n c o u r a g ea r e a c t i o nt oo c c u rb e t w e e nt h e m( B ) b i n d i n go f s u b s t r a t e( C )e n z y m es t r a i n st h ero enzyme rearrangesbound substratee l e c t r o n si n t h e s u b s t r a t e ,m o l e c u l ef,o r c i n gi tc r e a t i n gp a r t i a ln e g a t i v etoward a transitiona n d p o s i t i v ec h a r g e sstate to favor a reactionthat favor a reaction3-524.).As we saw for lysozyme, the active site of an enzyme contains preciselypositioned atoms that speed up a reaction by using charged groups to alter thedistribution of electrons in the substrates (Figure 3-528).
In addition, when asubstrate binds to an enzyme, bonds in the substrate often bend, changing thesubstrate shape.These changes,along with mechanical forces, drive a substratetoward a particular transition state (Figure 3-52C). Finally, like lysozyme, manyenzymes participate intimately in the reaction by briefly forming a covalentbond between the substrate and a side chain of the enzyme. Subsequent stepsin the reaction restore the side chain to its original state, so that the enzymeremains unchanged after the reaction (seealso Figure 2-22).TightlyBoundSmallMoleculesAdd ExtraFunctionsto ProteinsAlthough we have emphasized the versatility of proteins as chains of aminoacids that perform different functions, there are many instances in which theamino acids by themselves are not enough.
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