The mechanism, by which transhydrogenase couples transfer of H-equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane, has been investigated in the solubilised, purified enzyme from Escherichiu coli using analogues of the nucleotide substrates. The key observation was that, at low pH and ionic strength, solubilised transhydrogenase catalysed the very rapid reduction of acetylpyridine adenine dinucleotide (an analogue of NAD+) by NADH, but only in the presence of either NADP' or NADPH. This indicates that the rates of release of NADP' and NADPH from their binary complexes with the enzyme are slow. The dependences on pH and salt concentration suggest that (a) release of both NADP' and NADPH are accompanied by the release of H' from the enzyme and (b) increased ionic strength decreases the value of the pK, of the group responsible for H' release. Modification of the enzyme with N,Wdicyclohexylcarbodiimide led to inhibition of the rate of release of NADP' and NADPH from the enzyme, but had a much smaller effect on the binding and release of NAD', NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates.It is considered that the binding and release of H' , which accompany the binding and release of NADP+/NADPH, might be central to the mechanism of proton translocation by the enzyme in its membrane-bound state.

Proton-translocating transhydrogenase was solubilised and purified from membranes of Escherichia coli. Consistent with recent evidence [Hutton, M., Day, J., Bizouarn, T. and Jackson, J.B. (1994) Eur. 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-2H]NADH replacing [4A-1H]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 slower with [4B-2H]NADPH than with [4B-1H]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.

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