Lecture-24. Enzyme kinetics and Enzyme inhibition-ii

Transcription

1 Lecture-24 Enzye knetcs and Enzye nhbton-ii Noncopette Inhbton A noncopette nhbtor can bnd wth enzye or wth enzye-substrate coplex to produce end coplex. Hence the nhbtor ust bnd at a dfferent ste fro the substrate bndng ste. These nhbtors can destroy the catalytc actty of the enzye. The conforatonal changed happened due to bndng at dfferent ste of the substrate bndng ste, affect the catalytc ste. The stuaton for a sple sngle-substrate reacton wll be as follows: +S E ES P -S -I +I -I +I EI +S -S ESI Een ths s a coplex stuaton, for ES can be arred at alternate routes, akng t possble for an expresson of the sae for as the Mchaels-Menten equaton to be dered usng the general steady-state-assupton. Howeer, types of equaton noncopette nhbton consstent wth a Mchaels-Menten type and a Lneweaer-Burk plot f the equlbru assupton s ald. In the splest possble odel, sple lnear non-copette nhbton, the substrate does not affect nhbtor bndng. Under these E+I EI ES+I ESI condtons the reactons and hae an dentcal dssocaton constant, agan called as nhbton constant. The total enzye concentraton s effectely reduced by the nhbtor, decreasng the alue of but not affect on the alue, snce nether nhbtor nor substrate affects the bndng of each other. Let us agan dere an ntal elocty equaton, ths te we consder the specal case where s. As before, S] [ ES] In the presence of a non-copette nhbtor whch wll bnd equally well to E or to ES,.e. where [ E [ ES [ E ] [ E] + [ E + [ ES [ E] + + ([ E ] )( + )

2 [ E ] [ E] [ E ] [ E] [ ES] If we contnue wth preous equaton then we conclude that under these condtons: ( + ) Ths s the for of the Mchaels-Menten equaton, wth dded by a factor. Thus for the sple lnear non-copette nhbton, s unchanged but s decreased. So that, or Here s the alue of n presence of a concentraton [ of non-copette nhbtor. We get that for such a syste s the nhbtor concentraton whch hales the alue of. The Lneweaer-Burk equaton for sple lnear non-copette nhbton s: + And Lneweaer-Burk plots showng the effect of such nhbton are shown n the followng fgure. / / + Non-copette nhbtor slope slope / / Wthout nhbtor / ] 3 [ 3 [ 2 [ [I > [ 2 > [ /[S] /[S]

3 Once the type of nhbton has been establshed, the nhbton constant deterned usng the secondary plots. Snce And slope of the prary Lneweaer-Burk plot s It follows that plots of lnear, the ntercept on the X axs gng ay be / aganst [ and of slope the prary plot aganst [ are /' slope slope slope of prary plot / [ [ / A Dxon plot ay also be used to deterne. Fro Lnerweaer equaton we can wrte that + [ + + ( + + ) + + Thus a Dxon plot of / aganst [wll be lnear at fxed [E] and [S] for sple lnear non-copette nhbton. When, at that te t can be obsered that /. Hence the ntercept at the [ axs wll ge.

4 [S] / [S] 2 [ Soe enzyologsts ponted that sple lnear non-copette nhbton s the specal case and other fors of non-copette nhbton ay show dfferent characterstcs. Howeer, t s better to deterne the nhbtor pattern than the actual echans by whch the nhbton s gong on. It has becoe establshed that the nhbton pattern s establshed on the bass of ther actual nhbton pattern. On the bass, non-copette nhbton s only sad when the characterstcs of sple lnear non-copetee nhbton were obsered fro the Lneweaer-Burk plot. That s only s altered and no change on alue. Few clear cut nstances of noncopette nhbton of sngle substrate enzye catalyzed reacton are known. Major green tea polyphenols: epgallocatechn (EGC), epcatechn(ec), catechn, epcatechn gallate (ECG) and epgallocatechn gallate (EGCG) are establshed by Swagata Dasgupta s group as non-copette nhbtors RNase A. Heay etal ons and organc olecules whch bnd to SH groups of cysten resdues n the enzye are soetes quoted as beng exaple of noncopette nhbtors. Also there are soe other organc olecules and sall lgands whsh are known to bnd wth etal ons of enzyes and destroy the enzyatc actty. Howeer n such cases the olecules are behae as noncopette nhbtor n the Lneweaer-Burk plot. But as the olecules are bnd rreersbly rulng out the non-copette nhbton whch ust be reersble. Neertheless t should be understood that the nhbtors lke cyande, carbon onoxde, hydrogen sulfde, heay etal ons nhbted the enzyes to ther acton aganst enzye, whateer ther precse echans. UNCOMPETITIVE INHIBITION Uncopette nhbtors bnd only wth the enzye-substrate coplex not wth the free enzye. Substrate-bndng can cause a conforatonal change to take place n the enzye whch helps the nhbtor to bnd at nhbtor bndng ste or nhbtor can bnd wth the substrate bound enzye coplex. As the nhbtor not bnd to the substrate bndng ste, so the nhbton can not be oercoe by ncreasng the substarte concentraton. Both and are changed and a dstnct knetc pattern obsered under steady state condtons. The splest scheatc representaton as follows:

5 E +S -S ES E+P -I +I ESI Where ESI s the dead end coplex; the nhbtor constant Under steady-state condtons, S] [ ES] For ths syste, [ E ] [ E] + [ ES [ E] + [ E ] ( + ) [ E ] [ E ] [ ES]( + ) [ ES Now f we substtuted [E] n the preous equaton. The followng expresson we wll get S [ ] ( + ) + Now denonator and nuerator are dded by ges: [ S] + Ths s an equaton slar for of the Mchaels-Menten equaton, the constant both are dded by a factor. Thus for uncopette nhbton, and

6 and Here s the alue of n presence of concentraton [ of uncopette nhbtor and s the apparent alue of n presence of the sae concentraton of nhbtor. An nhbtor concentraton equal to wll hale the alues of and. The expresson of Lnweaer-Burk plot for the uncopette nhbtor s: + And the slope of the Lneweaer-Burk plot s: + Uncopette nhbtor [3 / Slope wthout nhbtor Slope / [2 [ [3>[2>[ /[S] /[S] That eans n the presence of the nhbtor for uncopette equaton the slope of the Lneweaer-Burk plot s not changed but the ntercept at X and Y axs s changed. The plots n presence of nhbtor are parallel wth plot n absence of nhbtor. The nhbton constant can be deterned usng the secondary plots. For uncopette nhbton we get the followng equatons.

7 and Hence plots of and (obtaned fro the prary plots) aganst [ are lnear and the ntercept at X axs s the alue of. /' /' [ [ Uncopette nhbton for a sngle substrate enzye nhbton s rare phenoenon; one of the exaples s nhbton of arylsulphatase by hydrazne. Howeer, uncopette nhbton pattern are obsered wth the two substrate reactons and ths wll help to elucdate the reacton echans. If a sple lnear Lneweaer-Burk plot obtaned but the plot s not atched wth the copette, non-copette or uncopette reacton then the nhbton process s called as xed type of nhbton for lnear nhbton.