Determination of Pre-Steady-State Rate Constants on the Escherichia coli Pyruvate Dehydrogenase Complex Reveals That Loop Movement Controls the Rate-Limiting Step

Spectroscopic identification and charicterization Of covalent and noncovalent intermediates on large enzyme complexes is an exciting and challenging area of modern enzymology. The Escherichia colt pyruvate dehydrogenase: multienzyme complex (PDHc), consisting of multiple copies of enzymic components and coenzymes, performs the oxidative 'decarboxylation of pyruvate to acetyl-CoA and is central to carbon metabolism linking glycolysis to the Krebs cycle. On the basis of earlier studies, we hypothesized that the dynamic regions of the Elp component, which undergo a disorder order transition upon substrate binding to thiamin diphosphate (ThDP), play a critical role in modulation of the catalytic cycle of pDHc. To test our hypothesis, we kinetically characterized ThDP-bound covalent intermediates on the Elp component; and the lipoamide-bound:coyalent intermediate on. the E2p component in PDHc and in its variants with disrupted active stie loops. Our results suggest that formation of the first covalent: predecarboxylation intermediate, C2 alpha-lactylthiamin diphosphate (LThbp), is rate limiting for the Seriea of steps culminating in acetyl-CoA formation. Substitutions in the active center loops produced variants with up to 900 fold lower rates of formation of the LThDP, demonstrating that these perturbations directly affected covalent catalysis This rate was rescued by up. to 5-fold upon assembly to PDHc Of the E401K variant. The Elp loop dynamic control: covalent catalysis with ThDP and are modulated by PDHc assembly, presumably by selection of Catalytically Competent loop conformations This mechanism could be a general feature of 2-oxoacid dehydrogenase complexes because such interfacial dynamic regions are highly conserved.