Conformational Ensemble Modulates Cooperativity in the Rate-determining Catalytic Step in the E1 Component of the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex

Sachin Kale,Frank Jordan

doi:10.1074/jbc.m109.065508

Sachin Kale, Frank Jordan

Open Access

https://doi.org/10.1074/jbc.m109.065508

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Abstract

Cooperativity is extensively used by enzymes, particularly those acting at key metabolic branch points, to "fine tune" catalysis. Thus, cooperativity and enzyme catalysis are intimately linked, yet their linkage is poorly understood. Here we show that negative cooperativity in the rate-determining step in the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex is an outcome of redistribution of a "rate-promoting" conformational pre-equilibrium. An array of biophysical and biochemical studies indicates that non-catalytic but conserved residues directly regulate the redistribution. Furthermore, factors such as ligands and temperature, individually or in concert, also strongly influence the redistribution. As a consequence, these factors also exert their influence on catalysis by profoundly influencing the pre-equilibrium facilitated dynamics of communication between multienzyme components. Our observations suggest a mode of cooperativity in the E1 component that is consistent with the dynamical hypothesis shown to satisfactorily explain cooperativity in many well studied enzymes. The results point to the likely existence of multiple modes of communication between subunits when the entire class of thiamin diphosphate-dependent enzymes is considered.

Highlights

Grant GM 50380. □S The on-line version of this article contains supplemental Tables S1 and S2 and Figs
The x-ray structures of the apo-E1ec [7], E1ec with thiamin diphosphate (ThDP) bound [7], or E1ec with C2␣-phosphonolactyl-ThDP bound (PLThDP; a stable analogue of the predecarboxylation covalent intermediate of ThDP formed with the substrate analogue methyl acetylphosphonate (MAP)) [8], and of apo-E1ec and E1ec variants complexed with ThDP and PLThDP [9] revealed no structural inequivalence of the active centers and provided no hint as to how the two sites communicate structurally
Allosteric communication between distant sites is fundamental to the catalytic functions of enzymes, allosteric communication linked to catalytic turnover is poorly understood [11]

Summary

Introduction

Grant GM 50380. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. We provide experimental evidence that the dynamics of both active center loops exhibit synchronous pre-equilibrium that occurs on a time scale similar to that of the rate-limiting catalytic step, and is essential for catalysis.

Results

Conclusion