PREDICTION AND SITE-SPECIFIC MUTAGENESIS OF RESIDUES IN TRANSMEMBRANEALPHA-HELICES OF PROTON-PUMPING NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASES FROM ESCHERICHIA-COLI AND BOVINE HEART-MITOCHONDRIA

Nicotinamide nucleotide transhydrogenase from bovine heart consists of a single polypeptide of 109 kD. The complete gene for this transhydro genase was constructed, and the protein primary structure was determin ed from the cDNA. As compared to the previously published sequences of partially overlapping clones, three residues differed: Ala598 (previo usly Phe), Val777 (previously Glu), and Ala782 (previously Arg). The E scherichia coli transhydrogenase consists of an alpha subunit of 52 kD and a beta subunit of 48 kD. Alignment of the protein primary structu re of the bovine trashydrogenase with that of the transhydrogenase fro m E. coli showed an identity of 52%, indicating similarly folded struc tures. Prediction of transmembrane-spanning alpha-helices, obtained by applying several prediction algorithms to the primary structures of t he revised bovine heart and E. coli transhydrogenases, yielded a model containing 10 transmembrane alpha-helices in both transhydrogenases. In E. coli transhydrogenase, four predicted alpha-helices were located in the alpha subunit and six alpha-helices were located in the beta s ubunit. Various conserved amino acid residues of the E. coli transhydr ogenase located in or close to predicted transmembrane alpha-helixes w ere replaced by site-specific mutagenesis. Conserved negatively charge d residues in predicted transmembrane alpha-helices possibly participa ting in proton translocation were identified as beta Glu82 (Asp655 in the bovine enzyme) and beta Asp213 (asp787 in the bovine enzyme) locat ed close to the predicted alpha-helices 7 and 9 of the beta subunit. b eta Glu82 was replaced by Lys or Gin and beta Asp213 by Asn or His. Ho wever, the catalytic as well as the proton pumping activity was retain ed. In constrast, mutagenesis of the conserved beta His91 residue (His 664 in the bovine enzyme) to Ser, Thr, and Cys gave an essentially ina ctive enzyme. Mutation of alpha His450 (corresponding to His481 in the bovine enzyme) to Thr greatly lowered catalytic activity without abol ishing proton pumping. Since no other conserved acidic or basic residu es were predicted in transmembrane alpha-helices regardless of the pre diction algorithm used, proton translocation by transhydrogenase was c oncluded to involve a basic rather than an acidic residue. The only co nserved cysteine residue, beta Cys260 (Cys834 in the bovine enzyme), l ocated in the predicted a-helix 10 of the E. coli transhydrogenase, pr eviously suggested to function as a redox-active dithiol, proved not t o be essential, suggesting that redox-active dithiols do not play a ro le in the mechanism of transhydrogenase.