THE INVOLVEMENT OF NADP(H) BINDING AND RELEASE IN ENERGY TRANSDUCTION BY PROTON-TRANSLOCATING NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE FROM ESCHERICHIA-COLI

Proton-translocating transhydrogenase was solubilised and purified from membranes of Escherichia coli. Consistent with recent evidence [Hutton, M., Day, J., Bizouam, T. and Jackson, J.B. (1994) fur. J. Biochem. 219, 1041-1051], at low pH and salt concentration, the enzyme catalysed rapid reduction of the NAD(+) analogue AcPdAD(+) by a combination of NADH and NADPH. At saturating concentrations of NADPH, the dependence of the steady-state rate on the concentrations of NADH and AcPdAD(+) indicated that, with respect to these two nucleotides, the reaction proceeds by a ping-pong mechanism. High concentrations of either NADH or AcPdAD(+) led to substrate inhibition. These observations support the view that, in this reaction, NADP(H) remains bound to the enzyme: AcPdAD(+) is reduced by enzyme-bound NADPH, and NADH is oxidised by enzyme-bound NADP(+), in a cyclic process. When this reaction was carried out with [4A-H-2]NADH replacing [4A-H-1]NADH, the rate was decreased by 46%, suggesting that the H- transfer steps are rate-limiting. In simple 'reverse' transhydrogenation, the reduction of AcPdAD(+) was 46% slower with [4B-H-2]NADPH than with [4B-H-1]NADPH when the reaction was performed at pH 8.0, but there was no deuterium isotope effect at pH 6.0. This indicates that H- transfer is rate-limiting at pH 8.0 and supports our earlier suggestion that NADP(+) release from the enzyme is rate-limiting at low pH. The lack of a deuterium isotope effect in the reduction of thio-NADP(+) by NADH at low pH is also consistent with the view that NADPH release from the enzyme is slow under these conditions. A steady-state rate equation is derived for the reduction of AcPdAD(+) by NADPH plus NADH, assuming operation of the cyclic pathway. It adequately accounts for the pH dependence of the enzyme, for the features described above and for kinetic characteristics of E. coli transhydrogenase described in the literature.