Kinetic resolution of the reaction catalysed by proton-translocating transhydrogenase from Escherichia coli as revealed by experiments with analogues of the nucleotide substrates.
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 Escherichia 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 pKa of the group responsible for H+ release. Modification of the enzyme with N,N1-dicyclohexylcarbodiimide 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.
Hutton
M
M
School of Biochemistry, University of Birmingham, England.
Day
J M
JM
Bizouarn
T
T
Jackson
J B
JB
D001665:Binding Sites; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011522:Protons; D012995:Solubility
D011522:Protons; D009243:NAD; D009249:NADP; D004024:Dicyclohexylcarbodiimide; C005941:3-acetylpyridine adenine dinucleotide; D009250:NADP Transhydrogenases