Dynamics of NADH Cofactor Binding to B. Stearothermophilus Lactate Dehydrogenase

Abstract

Laser induced temperature jump relaxation spectroscopy was used to study NADH nucleotide cofactor binding to lactate dehydrogenase from Bacillus Stearothermophilus thermophylic bacteria (bsLDH). The tryptophan fluorescence T-jump kinetics, that are determined almost exclusively by NADH binding/unbinding, show only one exponential relaxation component, while the NADH fluorescence T-jump kinetics, that report on NADH (un)binding and conformation changes around the active site, display three distinct relaxation components. The mid-range NADH relaxation rates are very close to the relaxation rates of the tryptophan kinetics, and their dependence on the total concentration of free ligand and enzyme shows a sharp initial increase and much slower rise at higher concentrations. The slow NADH relaxation rates are nearly independent of the total free ligand and enzyme concentration. Our kinetic data can be best described by a model in which NADH binding to the binding pocket of bsLDH is followed by a conformation change, presumably closing of the active site loop, and additional weak NADH binding to the binary complex bsLDH·NADH in the open-loop conformation only. The presumed open and closed loop conformations, that can be considered as competent and incompetent for further substrate binding, interconvert with relaxation time of about 1 ms.