Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex

TitleEfficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex
Publication TypeJournal Article
Year of Publication2008
AuthorsKale, S, Ulast G, Song JY, Brudvig GW, Furey W, Jordan F
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Pagination1158-1163
Date PublishedJan
Type of ArticleArticle
ISBN Number0027-8424
Accession Numberhttp://apps.isiknowledge.com/InboundService.do?Func=Frame&product=WOS&action=retrieve&SrcApp=EndNote&Init=Yes&SrcAuth=ResearchSoft&mode=FullRecord&UT=000252873900015
KeywordsACTIVE-CENTER, BINDING-SITES, COOPERATIVITY, coupling of dynamics to catalysis, E1 SUBUNIT, ENZYME, EPR, mobile loop dynamics, NEGATIVE, NMR, NMR CHARACTERIZATION, pyruvate dehydrogenase, REDUCTIVE ACETYLATION, SIDE-CHAIN ENTROPY, SYK KINASE, THIAMIN DIPHOSPHATE
Abstract

Protein motions are ubiquitous and are intrinsically coupled to catalysis. Their specific roles, however, remain largely elusive. Dynamic loops at the active center of the Ell component of Escherichia coli pyruvate dehydrogenase multienzyme complex are essential for several catalytic functions starting from a predecarboxylation event and culminating in transfer of the acetyl moiety to the E2 component. Monitoring the kinetics of Ell and its loop variants at various solution viscosities, we show that the rate of a chemical step is modulated by loop dynamics. A cysteine-free Ell construct was site-specifically labeled on the inner loop (residues 401-413), and the EPR nitroxide label revealed ligand-induced conformational dynamics of the loop and a slow "open <-> close" conformational equilibrium in the unliganded state. An F-19 NMR label placed at the same residue revealed motion on the millisecond-second time scale and suggested a quantitative correlation of Ell catalysis and loop dynamics for the 200,000-Da protein. Thermodynamic studies revealed that these motions may promote covalent addition of substrate to the enzyme-bound thiamin diphosphate by reducing the free energy of activation. Furthermore, the global dynamics of Ell presumably regulate and streamline the catalytic steps of the overall complex by inducing an entirely entropic (nonmechanical) negative cooperativity with respect to substrate binding at higher temperatures. Our results are consistent with, and reinforce the hypothesis of, coupling of catalysis and regulation with enzyme dynamics and suggest the mechanism by which it is achieved in a key branchpoint enzyme in sugar metabolism.

URLhttp://apps.isiknowledge.com/InboundService.do?Func=Frame&amp;product=WOS&amp;action=retrieve&amp;SrcApp=EndNote&amp;Init=Yes&amp;SrcAuth=ResearchSoft&amp;mode=FullRecord&amp;UT=000252873900015
Alternate JournalProc. Natl. Acad. Sci. U. S. A.