lecture 14 - 14-1MCB 354Lecture 14Bisubstrate...

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Unformatted text preview: 14-1MCB 354Lecture 14Bisubstrate ReactionsEnzyme Mechanisms: Chymotrypsin andLysozyme as Case StudiesBisubstrate reactionse.g. involving transfer of functional group from one substrate to the other: ATP + GlucoseADP + Glucose-6-phosphateIn general: A+ BP+ QhexokinaseIf 2 substratesand 2 products,called BiBiBiBireactions can be studied using Michaelis-Mentenapproaches:Can determine Kmfor each substrate (Aor B) by varying eg. [A]in presence of saturating [B]2 types:i)Sequential:both substratesbind to Ebeforeany productreleasedordered: obligatory order of addition of substrates and release of productsrandom: no obligatory order of binding & releaseii)Ping-pong:a productis releasedbefore all substratesare bound,and a functional group can transiently become covalently bound to E14-22. RandomEither Aor Bcan bind Efirst, then the other substrate binds, giving (EAB) complex. Aand Bare converted to products Pand Qon (EAB), giving (EPQ) complex. Then, either Por Qcandissociate from (EPQ), leaving EQand EP, from which P and Q then dissociate,regenerating E.SequentialBiBireactions1. OrderedSubstrate Abinds Efirst, giving EAcomplex. Then Bbinds EA, giving TERNARYcomplex(EAB). Aand Bare converted to products Pand Qon (EAB), giving (EPQ) complex.Pdissociates first from (EPQ), then Qfrom EQ, regenerating E.TERNARYCOMPLEXCLELANDS SHORTHANDNOTATION FOR BiBiREACTIONS:STATES OFENZYME(FREE OR INCOMPLEX)14-3Ping-pong reaction: covalentlymodified enzyme = intermediateFirst substrate Abinds to E (formingEAfirst), anda functional group is transferred from Ato Egivinga complex of modified enzyme Fand first product,P(FP). Pdissociatesfrom FP, leaving free F.Second substrate Bbinds F (forming complex FB),and Fs functional group is transferred from Fto Bto form second product Q, with reversion of Fto E.Resulting complex EQdissociates releasing Qand free E.14-4_1_[A]increasing [B][Can distinguish between random and orderedsequential mechanisms by the type of inhibitionby products Pand Q] Distinguishing betweensequentialand ping-pongreactionsby initial velocity experimentsSequential(both types)...
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This note was uploaded on 01/29/2012 for the course MCB 354 taught by Professor Spees during the Spring '09 term at University of Illinois, Urbana Champaign.

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lecture 14 - 14-1MCB 354Lecture 14Bisubstrate...

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