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.