SITE-DIRECTED MUTAGENESIS OF THE PROTON-PUMPING PYRIDINE-NUCLEOTIDE TRANSHYDROGENASE OF ESCHERICHIA-COLI

The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD(+) and NADP(+) coupled to the translocation of protons across the cytoplasmi c membrane. It is composed of two subunits (alpha, beta) organized as an alpha(2)beta(2) tetramer. This brief review describes the use of si te-directed mutagenesis to investigate the structure, mechanism and as sembly of the transhydrogenase. This technique has located the binding sites for NAD(H) and NADP(H) in the alpha and beta subunits, respecti vely. Mutagenesis has shown that the cysteine residues of the enzyme a re not essential for its function, and that inhibition of the enzyme b y sulfhydryl-specific reagents must be due to perturbation of the thre e-dimensional structure. The sites of reaction of the inhibitors N,N'- dicyclohexylcarbodiimide and N-(1-pyrene)maleimide have been located. Selective mutation and insertion of cysteine residues followed by cupr ic o-phenanthrolinate-induced disulfide crosslinking has defined a reg ion of interaction between the alpha subunits in the holoenzyme. Deter mination of the accessibility of selectively inserted cysteine residue s has been used to determine the folding pattern of the transmembrane helices of the beta subunit. Site-directed mutagenesis of the transmem brane domain of the beta subunit has permitted the identification of h istidine, aspartic acid and asparagine residues which are part of the proton-pumping pathway of the transhydrogenase. Site-directed mutagene sis and amino acid deletions have shown that the six carboxy terminal residues of the alpha subunit and the two carboxy terminal residues of the beta subunit are necessary for correct assembly of the transhydro genase in the cytoplasmic membrane. (C) 1998 Elsevier Science B.V.