Effects of metal ions on the substrate-specificity and activity of proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli.
Nicotinamide nucleotide transhydrogenase catalyzes the reversible reduction of NADP+ by NADH and a concomitant proton translocation. It was demonstrated (Glavas, N.A. and Bragg, P.D. (1995) Biochim. Biophys. Acta 1231, 297-303) that the Escherichia coli transhydrogenase also catalyzed a reduction of the NAD-analogue 3-acetylpyridine-NAD+ (AcPyAD+) by NADH at low pH and in the absence of (added) NADP(H) and high salt concentrations The mechanism of this reaction has as yet not been explained. In the present study, the E. coli transhydrogenase was purified by affinity chromatography through the NADP(H)-site, rendering the pure enzyme free of NADP(H). Using this preparation it was confirmed that the enzyme readily catalyzes the above reaction. Inhibitors specific for the NADP(H)-site, e.g., palmitoyl-Coenzyme A and adenosine-2'-monophosphate-5'-diphosphoribose, strongly inhibited the reduction of AcPyAD+ by NADH, whereas an inhibitor of the NAD(H)-site, adenosine 5'-diphosphoribose, was less inhibitory. This suggests that a lack of metal ions or other ions at low pH induces an unspecific interaction of the NADP(H)-site with AcPyAD+ or NADH, presumably NADH, producing a cyclic reduction of AcPyAD+ by NADH via NAD(H) bound in the NADP(H) site. A stimulation of reduction of AcPyAD+ by NADPH by Mg2+ present during reconstitution of transhydrogenase in phospholipid vesicles was observed, but it is presently unclear whether this effect is related to that seen with the detergent-dispersed enzyme.
Zhang
J
J
Department of Biochemistry and Biophysics, Chalmers University of Technology, Göteborg, Sweden.
Hu
X
X
Osman
A M
AM
Rydström
J
J
D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D009994:Osmolar Concentration; D010171:Palmitoyl Coenzyme A / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity
D008670:Metals; D011994:Recombinant Proteins; D012492:Salts; D015636:Magnesium Chloride; D009243:NAD; D010171:Palmitoyl Coenzyme A; D009249:NADP; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D009250:NADP Transhydrogenases