THE INVOLVEMENT OF NADP(H) BINDING AND RELEASE IN ENERGY TRANSDUCTIONBY PROTON-TRANSLOCATING NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE FROMESCHERICHIA-COLI

Proton-translocating transhydrogenase was solubilised and purified fro m membranes of Escherichia coli. Consistent with recent evidence [Hutt on, M., Day, J., Bizouam, T. and Jackson, J.B. (1994) fur. J. Biochem. 219, 1041-1051], at low pH and salt concentration, the enzyme catalys ed rapid reduction of the NAD(+) analogue AcPdAD(+) by a combination o f NADH and NADPH. At saturating concentrations of NADPH, the dependenc e of the steady-state rate on the concentrations of NADH and AcPdAD(+) indicated that, with respect to these two nucleotides, the reaction p roceeds by a ping-pong mechanism. High concentrations of either NADH o r AcPdAD(+) led to substrate inhibition. These observations support th e view that, in this reaction, NADP(H) remains bound to the enzyme: Ac PdAD(+) is reduced by enzyme-bound NADPH, and NADH is oxidised by enzy me-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 b y 46%, suggesting that the H- transfer steps are rate-limiting. In sim ple 'reverse' transhydrogenation, the reduction of AcPdAD(+) was 46% s lower 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 su pports 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 t he view that NADPH release from the enzyme is slow under these conditi ons. A steady-state rate equation is derived for the reduction of AcPd AD(+) by NADPH plus NADH, assuming operation of the cyclic pathway. It adequately accounts for the pH dependence of the enzyme, for the feat ures described above and for kinetic characteristics of E. coli transh ydrogenase described in the literature.