Structural and functional studies of the pyridine nucleotide transhydrogenase of Escherichia coli

The genes for the E. coli transhydrogenase enzyme have been cloned and sequenced in this lab and overexpressed in the membranes of E. coli (Clarke et al., 1986, Eur. J. Biochem. 158, 647-653). The E. coli transhydrogenase was found to consist of an α subunit (54588 Da) and a β subunit (48691 Da) arranged as an α₂β₂ dimer (Hou et al., 1990, Biochim. Biophys. Acta 1018, 61-66). The transhydrogenase enzyme was studied with respect to topology, location of the active sites, mechanism of proton pumping and mechanism of hydride transfer. The transhydrogenase was purified from E. coli membranes overexpressed with this enzyme as a soluble or a membrane-bound preparation. The soluble transhydrogenase was able to catalyze hydride transfer between ApNAD+ (3-acetylpyridine adenine dinucleotide) and NADPH, while in the membrane-bound transhydrogenase, this reaction was linked to the translocation of protons. The structure of the transhydrogenase was probed by limited trypsin digestion of both soluble and membrane-bound preparations. N-terminal amino acid sequences were obtained from the resulting fragments. These results led to a topological model of transhydrogenase in the membrane to be constructed. NADP+ and NADPH were found to introduce a conformational change in the β subunit resulting in two additional fragments derived from the β subunit upon trypsin digestion. Since transhydrogenase is known to contain separate binding sites for NAD(H) and NADP(H), the location of these was examined by covalent modification. FSBA (5'-pfluorosulfonylbenzoyladenosine) and DCCD (N,N'-dicyclohexylcarbodiimide) were both found to label near the NAD(H) binding site in the a subunit at aY226 and aD232,E238,E240 respectively. As well FSBA labelled another site in the p subunit at pY431, while DCCD labelled the transmembrane domain of the p subunit. The other site of FSBA labelling was proposed to be at the NADP(H) binding site. A residue βG314, when mutated, was found to abolish transhydrogenase catalytic activity as well as the NADP(H)-induced conformational change ability of the p subunit as probed by trypsin digestion. The sequence around this residue suggested the presence of another NADP(H) binding site on the p subunit. DCCD labelling followed by measurement of hydride transfer and proton translocation activities of wild-type transhydrogenase as well as a mutant where DCCD only labelled the transmembrane domain of the β subunit has shown that these two activities are coupled. The distance of DCCD labelling from the surface of the membrane was studied using NCD-4 (Ncyclohexyl- N'-[4-(dimethylamino)naphthyl]-carbodiimide), a fluorescent analog of DCCD, by quenching of the fluorescence with spin labels which intercalate into the membrane at various distances. The site of DCCD labelling in the transmembrane domain of the β has not been determined due to difficulty in isolating any sequencable peptide. Site-specific mutants of conserved residues in the transmembrane domains of the a and pp subunits were analyzed and PH91 was found to be implicated in proton translocation. A mutant, PH91N, demonstrated catalytic activity but this was not coupled to proton translocation activity. Therefore the two activities have become uncoupled in this mutant. The presence of two nucleotide binding sites on the β subunit in addition to the NAD(H) binding site on the α subunit was shown by affinity chromatography of the β subunit on NAD and NADP agarose columns, as well as by transhydrogenation between NADH and ApNAD+. In wild-type transhydrogenase, NADH reduced ApNAD+ only in the presence of NADP(H), but in mutants where the NADP(H)-induced cleavage of the p subunit had been disrupted so that p was cleaved in the absence of substrate, ApNAD+ was reduced by NADH in the absence of NADP(H). These experiments have demonstrated that there is a NAD(H) binding site on the a subunit and NADP(H) and NAD(H) binding sites on the β subunit and have given insight into the mechanism of hydride transfer.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat