Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli

Abstract

1. The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, CI and j, of apparent M , 50000 and 47000.2. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of the fluorescence of 9-aminoacridine.3. Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatory linked. NADH protected the enzyme against inhibition by N,N'-dicyclohexylcarbodiimide, while NADP, and to a lesser extent NADPH, appeared to increase the rate of inhibition. [ ''C]Dicyclohexylcarbodiimide preferentially labelled the 50 000-M, subunit of the transhydrogenase enzyme.4. The presence of an allosteric binding site which reacts with NADH, but not with reduced 3-acetylpyridine adenine dinucleotide, has been demonstrated.Pyridine nucleotide transhydrogenase, found in the cytoplasmic membrane of Escherichia coli and in the inner membrane of mitochondria, catalyzes the reversible transfer of a hydride ion equivalent between NAD and NADP. The transhydrogenation between NADH and NADP is coupled to respiration and ATP hydrolysis (see [I] for review).During the past decade, a large body of evidence has accumulated from studies on the mitochondria1 transhydrogenase to support Mitchell's hypothesis [2] that the transhydrogenase functions as a proton pump and translocates protons across the membrane according to the equation [31: nH; + NADPH + NAD nH&, + NADP + NADH .Comparatively little work has been done with the corresponding enzyme from E. coli despite advantages that genetic manipulation of this system can offer. The elucidation of the subunit composition and molecular mechanism of the E. coli transhydrogenase requires purification and reconstitution of the functional enzyme. So far the E. coli transhydrogenase has been only partially purified [4-61. SDS/ polyacrylamide gels of the preparation purified by Liang andCorrespondence to

Kinetic resolution of the reaction catalysed by proton‐translocating transhydrogenase from Escherichia coli as revealed by experiments with analogues of the nucleotide substrates

“…In contrast to an earlier study [29], (cHxN),C was found to react with both the a and p polypeptides of the enzyme, and we must therefore withdraw an interpretation of our experiments [32], which was based on that finding, that the (cHxN),Creactive group is located within 100 residues of the C-tenninus of the a polypeptide. Furthermore, we can now predict that some or all of the newly identified (cHxN),C label in the p polypeptide, which is enhanced by NADP(H) [39], will be associated with the inhibition of the rate of release of NADP' and NADPH from the enzyme.…”

“…The presence of either NADP' or NADPH during treatment increased the degree of inhibition [29]. We confirmed these observations (data not shown).…”

“…We had predicted [32] that this explanation might also account for some earlier observations that had been made with (cHxN),C-treated, solubilised and purified H+-transhydrogenase from E. coli [29]. The results described below indicate that this is indeed the case.…”

“…Treatment of purified H'-transhydrogenase from E. coli with (cHxN),C inhibited the rate at which the enzyme catalysed the reduction of AcPdAD' by NADPH [29]. The presence of either NADP' or NADPH during treatment increased the degree of inhibition [29].…”

“…8) and NADPH-dependent (data not shown) reduction of AcPdAD' by NADH. It was previously suggested [29] that the presence of NADH (but not NAD' or AcPdAD') during treatment of H+-transhydrogenase from E. coli with (cHxN),C protected against the inhibition of AcPdAD' reduction by NADPH. However, results of our experiments cast doubt over this conclusion (also see [32]).…”