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 cytoplasmic membrane. It is composed of two subunits (alpha, beta) organized as an alpha 2 beta 2 tetramer. This brief review describes the use of site-directed mutagenesis to investigate the structure, mechanism and assembly of the transhydrogenase. This technique has located the binding sites for NAD(H) and NADP(H) in the alpha and beta subunits, respectively. Mutagenesis has shown that the cysteine residues of the enzyme are not essential for its function, and that inhibition of the enzyme by sulfhydryl-specific reagents must be due to perturbation of the three-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 cupric o-phenanthrolinate-induced disulfide crosslinking has defined a region of interaction between the alpha subunits in the holoenzyme. Determination of the accessibility of selectively inserted cysteine residues has been used to determine the folding pattern of the transmembrane helices of the beta subunit. Site-directed mutagenesis of the transmembrane domain of the beta subunit has permitted the identification of histidine, aspartic acid and asparagine residues which are part of the proton-pumping pathway of the transhydrogenase. Site-directed mutagenesis 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 transhydrogenase in the cytoplasmic membrane.
Bragg
P D
PD
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada. pbragg@unixg.ubc.ca
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary
D009243:NAD; D009249:NADP; D009250:NADP Transhydrogenases