The kinetic mechanism of pyridine nucleotide transhydrogenase from Escherichia coli.

Pyridine nucleotide transhydrogenase was extracted from the membrane fraction of Escherichia coli with Triton X-100 and purified i’l-fold by chromatography on DEAE-cellulose and Sepharose 4B in buffers containing detergent. The kinetic mechanism for reduction of I-acetylpyridine adenine dinucleotide (AcPyAD) by NADPH was determined. Plots of reciprocal velocity versus the reciprocal concentration of either substrate at different fixed concentrations of the other substrate were intersecting, indicating a sequential mechanism. W-AMP was a competitive inhibitor versus AcPyAD and was noncompetitive uersus NADPH, whereas 2’-AMP was competitive uersus NADPH and noncompetitive uersus AcPyAD. These results establish that the mechanism is random. In product inhibition experiments, NADP was competitive with NADPH and noncompetitive versus AcPyAD; AcPyADH was competitive with AcPyAD and noncompetitive uersus NADPH. These patterns are consistent with a random mechanism in which dead-end complexes of E l NADPH l AcPyADH and E .NADP*AcPyAD can form. NAD, an alternative substrate, is competitive with AcPyAD with respect to AcPyADH production and noncompetitive with NADPH, which is also consistent with a random mechanism. In the reverse direction, reciprocal plots were concave downward. The effect of MgATP on the kinetics of transhydrogenase was studied with membrane preparations from E. coli. At 0.1 mM substrate concentrations, MgATP increased by at least 3-fold the rate of reduction of NADP and analogs by NADH and analogs. V,,,,, for reduction of NADP by NADH was increased 2.2-fold by MgATP. Reciprocal plots were concave downward, both in the presence and absence of MgATP.