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mechanisms_for_catalysis

mechanisms_for_catalysis - Mechanisms for Catalysis How do...

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Unformatted text preview: Mechanisms for Catalysis How do enzymes catalyse reactions? What are the different foreeefeffecta that they have at their disoposal for achieving rate enhancements of 1013 or more over reactions in solution? Can we explain it in terms of known chemicalfphysical priciples? Entropy and reduction of molecularity Remember from thermcdynamicaftranaiticn state theory that sci . 1131 - Thai m In order to bring two molecules together to make one in the transition state. substantial e1‘fl.‘.rcu;+}ur must be lcat. The entropy change, 5.31, then, is negative and this makes he: tend towards positive {i.e. the reaction is more difficult}. one obvioue_way to lower dB: and thereby increase the reaction rate is to bring the two reacting groups close together in the ground state by some teens, so that no large entrogy change is needed to get to the activated complex. Schematically, this looks like (I)H T“ 1 Cf—l large ‘55 A B + HZX 511111113155i [ii—I e @J’HX To get an idea of the magnitude of this effect we can look at some model chemical reactions. ---r 1H.|r| jun—gluin- lllnhnl III' man-l Mtg-fl acid “'9 ' * ' - L stages .. . _.__.—~ tam.- mn . . :l. 3 {our I. I L”. mom . . . _ m :- - l 2:: - fl The reaction on the left is the formation of an anhyflride from an ester and anfi acid. Here, we assume that both.the himolecular and unimoleoular reactions have similar heats of activation since the chemical groups it'nlrolnreol in bond; making and breaking are identical. So, the difference should be fine to the entropies of activation- We can get a feeling for this entropic actination by tethering the two reactants together by calculating an effective concentration of the mmflwgmwfmmflemficmmmm.hrmemmfimonmehfi this is effective [cog-1 = mtg = 2 x’ 11:5 s {g} This says that. simply by tethering the two carboxylate groups together on the same molecule. we have achieved a reaction rate that woulfi take 2 x l05 H free acetate to watch it. This is a Very large effectiVE concentration ineeee. The series of reactions shown on the right hand side of the figure shove go from a unimoleoular reaction with freeaom of rotation about the intervening bonds {3: to one that is rigifily restrained in the-reactive conformation {11. The effective molarity of the groups in 3 is fine M compared to the bimolecular reaction. It is clear that as we introcuce more conformational restraints into the molecule. the reactivity is greatly enhanced. The effective molarity of the groups in 1 is 3 x in? H compares to the bimclecular reaction. The carbonylete in this reaction is a general base catalyst and a water molecule occurs in the transition state. Were this not so. the effective concentration might be even larger. This entropic advantage is not free in these molecules. The entropic cost was paid for during the synthesis of the molecules with both reactive groups. Enzyme catalysed reactions also have to pay for this effect. In order to bring the molecules together on the enzyme surface. the binding energy between the enzyme and substrates is used to pay for the decrease in entropy. One very important point should be made here. The reactive groups must be brought together in a productive orientation. For example, you can imagine that with 6 above. if you measured the rate constant for the trans isomer it would be quite a bit smallerl The enzyme also has to bring reactive groups together in the correct orientation. In fact. you could imagine a protein inhibiting a reaction by binding reactants much too far away from each other. Covalent catalysis A second way that enzymes accelerate chemical reactions is that they provide completely new reaction pathways compared to those available to solution reactions. These new pathways are generally more complex, but the additional steps“are compensated by their ease. with the overall result that the reactions proceed much faster. First let's look at an example of electrophilic catalysis. Acetoacetate undergoes a spontaneous decarhoxylaticn reaction, as dc: all B keto acids. The carbanion left after C02 loss is stabilized by delocalisation into the carbonyl group. <3 W ”F CHICfC; —r CHJCX +1.20; Val o “a. he enzyme called acetoacetate decarboxylase catalyzes this same reaction. but by a more complex pathway. final/CH, n'l. H! CO: CH: H' s—ih-I. EO=C\ L 5—1;: -)—’ E—g— —* as C CH. H. a” ' cf; “0” Re 9/ o “ /CH: Enfi=c\ “=0 H' + e—NH. + carom-I3 con Here, the enzyme provides a lysine in the active site that forms a Schiff base with the Rate group. This protonated Echiff base is a much better electron sink than is the keto group itself and therefore the carhenionic intermediate is much lower in energy and easier to form. The enzymatic pathway includes two more steps than the one in solution but these are both quite facile. The overall result is a such greater reaction rate on the enzyme surface compared to solution. Many other enzymes use electrophilic catalytic mechanisms. These include all pyridcxal phosphate and thiamine diphosphate dependent enzymes. nucleophilic catalysis is also common in enzyme reactions. LookIr yet again. at the serine protease mechanism. The amide group-of the polypeptide chain is very unreactive and we need a good nucleophile to attack the carbonyl group because it is highly stabilized by the following resonance forms. {3} The serine proteases are so named because they use an active site serine as a nucleophile. This group is a better nucleophile than is water and it is additionally activated by the Asp—His pair in the active site. The mechanism is given helm. if h- an assign-ii” eel—Neil, is '“ E a .33“) . Janeen» an??? N {I H. I. TIMW V. 1w We. 1|, 1L §=\ 3'" i“ ‘9 "es?” (“in {Ia—HM __H_nr5""' t: I: a d a... *r ““ "5i my mean K ‘1 W .m I‘I'l. M ennui“- Hints-13 fimkmmmwfiflmfihflmliwm“ mmflhiflflmflmflfl. "1:.er Here, the activated serine hydroxyl group attacks the amide carbonyl and forms a tetrahedral intermediate, which kicks out the amine and gives a covalent acyl enzyme intermediate. This ester is then hydrolyzed giving the free enzyme and the acid. The key stop here is the activation of the serine hydroxyl by the enzyme for the nucleophilic attack on the amide carbonyl. Another example of nucleophilic catalysis is the reaction catalyzed by glyceraldehyde phosphate dehydrogenase. This enzyme catalyzes the oxidative phophorylation of glyceraldehyde phosphate to diphosphoglycerate. 'H' film KE;TW—ores ==IhEL—oheuEEEEHEEA—mpemu .'. E E T H-fifi—firiu ‘3 me +eeoeee == o . at ° 5..“ h woweu The key step in this reaction is the formation of the thiohemiacctal, which is readily oxidized by NED+ to the thioester. This ester; in turn; undergoes phosphorolysis to yield the final products. In this case, the enzyme thiol group is a much better nucleophile than is phosphate, which would first have to add to the carbonyl in an analogous solution reaction. Again. we see that the enzyme reaction is more complex. but the ease of the component steps gives a large overall increase in the rate constant for the reaction. General acidfbase catalysis It is difficult to think of an enzyme catalyzed reaction that does not involve at least one proton transfer step. Convince yourself by surveying mechanisms in Walsh's book IEnzymatic Reaction Mechanisms". General acidfhase catalysis :thtl works by allowing reactions involving proton transfers to proceed by mechanisms involving lower energy species than are possible in unoatalyzed reactions. This nacessarily involves charged species because general caidsihases donatefaccept a proton. which is a hard little hall of positive charge. The following reaction scheme shows three pathways for the dehydration of a carbinolamine; The upper pathway shows the ancatalyzed reaction; This involes the expulsion of DH“ from the transition state, which is a high energy species. The bottom pathway shows the specific acid catalyzed reaction. This involves 'the formation of the unstable cxonium ion, which. then eliminates water from the transition state. To the extents to which the transition states of these two pathways have the character of these unstable species, they will be destabilized and slow the reaction. The middle pathway shows the general acid catalyzed reaction. The key to the lower energy {and therefore larger rate constant] for this pathway is that no highly unstable species are formed. Both the acidic and basic forms of the catalyst are relatively stable. and the transition state has charge character only on species that handle it well. Thus, we see that GAB: provides reactions involving charged species alternate pathways that avoid highly unstable charged intermediates. The stabilization provided by the GARE is not simply of a hydrogen bonding character. In the middle ease pathway shown above, for example, the water molecule has oxonium ion {a strong acid} character from which proton transfer to the basic form of the catalyst is thermodynamically favorable. Here, the GARE involves, in a sense, a thermodynamic driving force for proton transfer in the transition state. There are two distinct groups of substrates for GABC: carbon acidsfbases {i.e. CH bonds being brokenfmade} and heteroatom acids {i.e. Rfl—H. RN+u H. RS-Hi. Proton transfer to and from heteroatcms is generally very fast, often diffusion controlled. It is a reasonable generalization to state that, in GAE: where the proton transfer is between heteroatoms only, the proton transfer rate itself is not rate limiting. In the case reaction above, the rate of breaking the NLU bond can be considered the limiting process.r which, of course, is helped along by the case. Carbon acids are a different story. Proton transfers to and from carbon are generally much slower than to and from heteroatnms. Why is this? Consider the initial and final species in the proton transfer reactions. B'+-HOH =E=== EH-+HEF 3‘ + HEH % EH + H5' B'+HN+H=";—-- BH-I-EN {41 In the case of heteroatoma there are no large structural rearrangements needed to stabilize the charged species because the heteroatoms are either electronegativeIr have a lone pair.r or the electrons reside in d orbitals {in the case of 5}. The conjugate bases cf carbon acids generally undergo electronic rearrangements in order to stabilize the negative charge by resonance interactions. For example, _._ :5: The large structural rearrangements necessary for resonance stabilization of earhanions to occur makes a substantial contribution to the large activation energies for proton transfer from carbon acids. 4F Diffusinn cuntrclled He 5. The mun bin-in- for the master of a proton hound mamas is mud: lure;- thln far one bound to mum. The bar; tie: in the litter and I: jut that in diffusion. so the barrier for the Mn h‘lnfl‘lr Ind! 1! film tfl-I Haas that far dEIuEIon. Reaction coordinate H—D— In the case of carbon acids. one can consider CHH bond breakage to be the primary event that determines the how fast the transition state is reached. we will find many examples of GAE: insclving carbon acids in biochemistry. The following tables present data showing the sensitivity to GARE of reactions of carbon acids of different acidity. These results are expected from the Hammond postulate. TIMI "1 llul'lhtu fig. 14" I"... flu,“ Tang 1.11. "in! II 1hr "HI! at the III‘II-n "H an Inn- mid-'- fifl- nun-M: Io menu's-“1...“ “mgr.“ Hum“ 'Fflflfl' 11'! to urn-runn- nah-I. r —-F———_———'—-———_— a WIT-r PK. dame . H Eli-II El! “I h ' II. .l '__T_‘_‘———*————---——-—-————_——____. a toss 4.55 sin in 33:. ‘3‘ arc-mm“ '5 u- $m¢s§mroos Lu: or: us as “33'5"” fl :5. u mousse «on 15.1. m DI— all! [HIE-ED I a mmn km 15.! '11! m- "4 Ens-s1: HI 4: BILGOCHCI: -—in us M: NH m _ 7‘” ':'*15‘*':'E'-'!3I ‘0 41' cement. Iil 4.1-5 $3.1 in cm' -. u C: Hum-Ii! nu- ml: 4 Egan. an aim in .. LEE} stew 3*: as" m == ' I- : . can an moms to as -J.ns' was as mflflflmm‘ 3.1 fl. Ell-IO . do a v. I ._ egos. o2 s‘f'm 3 3w magma m m w :3 -1.': ammo as: :2 LE". . Hm“ ‘4 EILEHD 4.1 u . "T marsh in 1: mlmcslcoeu, -m 9.: u: __—'———'-—————_____ (summon. as: is an . calms-toot. +11!- EJ M2 I"Fl. 1'. HIE. "Th Fem In. mm." y. 171.. Rind]. L‘fil‘tflkr Prll. Im.N.-Y.. nil. ”Pu-[build p.141]. 11-: N" we"- Conformational changes and the tee of binding energy in catalysis ‘ g. Substrates for enzymatic reactions are frequently quite large and present the enzyme with several functional groups and substantial hydophcbic surface with which favorable binding interactions can be made. If the enzyme made use of all possible interactions with the substrate for binding only. the observed dissociation constants could be very low indeed. This is illustrated by the femtomolar dissociation constant. between biotin and avidin [a biotin binding protein} . very tight binding of substrates by enzymes is not generally a good thing. It is generally accompanied by very low dissociation rate constants, which can limit the overall rate of an enzymatic reaction {as in the case of coenzyme dissociation from several dehydroganases} . Also. if the K}; value is much lower that the physiological concentration of the substrate, then the enzyme. will always be saturated, and one mechanism for controlling the rate of the net flux through a reaction in vivo is lost [i.e. rate goes try-{down with [5] when [S] is near KHJ. It is found experimentally the in vino [SI and KM are generally closely matched. For example. the Ed = 10'15 for avidin binding biotin is acheived by an association rate constant of '3 :5: ill7 M‘ls'l and a dissociation rate constant of 9 x lfl'a s'l. This gives a dissociation half life of a phenomenal 2.5 years! Here, WE introduce the concept of intrinsic binding energy. This is the free energy of interaction between. an enzyme and a. substrate if completely optimal interactions were made between enzyme and substrate without any strain incurred. This is illustrated in the diagram below. -———— "—‘—————__. f | +. Slrain energy - Observed Adrianne Blndlng Energy mndTng as 4} ( Iii-G Lrttrinslc H A E B BlndIng Ease | 11 EnergyI Available For A B catalytlc Won: The observed binding’anergy is the free energy corresponding to the observed KS-velue. The,fiifference between the intrinsic and observed binding energies is the energy available to the enzyme from potential ELS binding interactions that can be used to increase the reactivity of the E—S complex. either by ground state effects {shown} or transition state effects {not shown}. Here, we will discuss two quite different means by which the excess binding energy between enzyme and substrate can be used towards catalysis. The first is in the attainment of high substrate specificity. and the second is in the direct utilization of binding energy to lower the activation energy for chemical steps on the enzyme surface. The induced fit hypothesis states that the unbound enzyme is in equilibrium between active and inactive conformations. and that binding of the correct substrate induces the enzyme, by mass action; to go t1} the active conformation. In the example above. if the locus of reaction is on the "E“ part of the substratE, then a substrate without '1' would not react as fast because the extra binding energy needed to'close the enzyme into the reactive conformation. is .not. available. This is illustrated below. Fin 3. The induced-fit medIsm'sI'n. in which binding nfsnhmate through stones ‘3. and B, cum I. dim-Ire in the mint-madame? rh- merme so that the maps 3: and Y in the and?! sits. whim u: malted for catalysis. m properiy positioned relative to The mtsmhe.‘ The mechanistic scheme associated with.this mechanism is shown below. Iasct;:§== acts'____i K” Kir ”K. Ei-n ? Bin 3 Fersht (p. 332} has pointed out that this general scheme will not lead to an increase in substrate specificity for the correct substrate under some circumstances. This is because it is only the distorted enzyme that is active and this distortion takes energy. The essence of his arguments contain the assumption that the active enzyme can bind and release substrates just as well as the inactive enzyme. This argument is not valid for the case where only the inactive enzyme can bind and release substrates. This is not an uncommon situation. There are many examples of enzymes that undergo large conformational changes upon binding substrates. such that the substrates are fully {or nearly so} sequestered from solvent in the closed conformation of the enzyme. Examples include the enzymes hexokinase. citrate synthase, and aspartate aminotransferase. In these cases. the reactivity towards a particular substrate can be enhanced by a factor equal to the amount by which it can push the conformational equilibrium more towards the active form compared to another substrate. What is happening here is that the enzyme is using excess binding energy to achieve a high substrate specificity. The'enzyme uses some of the intrinsic binding energy to close domains together. for example, but it doesn't really need it for tighter binding. The direct utilisation of binding energy for catalytic work is a more debated consent. The idea is that excess substrate binding energy can be directly transduced into catalytic power by some mechanismEsJ. How can one imagine this? There are two general mechanisms differing in the identity of the complexes in which favorable. distal interactions are maximised. The first possibility is the strain mechanism. Here. the enzyme might compress two reactive groups together. using excess binding energy to drive the unfavorable steric interactions. An example of strain accelerating a chemical reaction is given below. h ._ CH: CH: C CH HG Cl a D 5' :1: éHI-I: ‘1. 5 J o 'E-‘fl—P—u tee HP“ \P” as / kirk" = "3' 1t] A specific enzymatic example of this might be the forcing of the Ber—DH of a serine protease up against the carbonyl group of a peptide substrate. distorting it towards the tetrahedral geometry of the adduct. The data presented below on elastase and {t—lyitc protease, serine proteases. is suggestiw of something like this. rate 9—: seem ran-mam fares Hydrolfrir ofPtplidt Eufirtrator by Two Relative N 1321' r: Eel-En: Prawn ELASTAS E u-L‘t'TIC PRGTL’LEE i=4 R... MK... is. K. UK mama-rt {5' *1 {null} {riu- *1. (r1: {ms-l: {ta-3.3:} Pa ‘P1 ‘1’: ‘P: '15: ff 'Pi mas—NH, saunas IED «canto «taunt-4 ' 3m {41.0013 At-Pro-Att-NH= ELEM. Ifla ELI}? DJ}? Ball] 13.23 films—fitl:al.v.'P'rI:I--.-’I.la-NHE DJIIEI 4.2 21 Duff! 11!) 5.5 AtvPro-Ala-Pro-Ala~NI-I= 3.5 3.9 22ml LI Ed 43 AC-fila-PrD-ME‘Fm—AII-NHE 5.3 5.9 IEEI} $9? [5 5-} At-Pro-Ala-Pmfily -NH! ELI 22 5 [1.13 $3 $9 Ht‘Plfl-AlarProi-Vll ail-1: E.“ 35 203 [It-£2 3'5 55 AoPro-Alaho—Lw-N HI 5.1! I l 270 {11.5% 159.9 {5.2 As13'ro—J‘tla-P'ro-r'tlatrlfilzr-Z‘~ZH= 25 4.3 55110 3.43 11? ESE Achro-Ma-Pro-Aia-AI: NH: 3? L5 247W 2.5? 5.5 50D At-Pro-AJa-Pwfila—icle-Ig IE [LE-I 233m LEE 5.9. 255 At—Pro-Ma-Pro-xflasfija-fla-EH: — — — 1?.5 5.3 2MB CI fit- i: :11: CHSEI— group blocking the machine group of the :1.th residue: —."~'H: is thl: amid: you? on tho u-arboxfl slip. These not demonstrate that WK»: increase: mgr Ina-Edd when [11: size of the subsu-att Estrada from asinglt ruiduc to a htxap-cptide. primal-ii? do: In inns-tel: in km. Interactions with substrate residue 17": in: most important for e-lylit protease. but with dost-as: i: is residue P4,. Elana" prefer; an A1: rcsidtn: at P... but o—lgoit pros-=51: toltmlu both smaller and slightly larger side chains. Intmfiinns with miduts F.’ and P; om. increase kmffim. but there i: lint: specificity For 1: amino acid side chain. This table Shel-J's several examples of much. greater increases in lac-1t than in KM for given changes in substrate structure. Look, for example. at the two pair of data for elastase that are marked with connected arrows. The tap pair Show a lDflD—fold increase in kc“; and only a 25-fold decrease in KM for the addition of two amino acids to the peptide subs...
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