Site-directed mutagenesis of the proton-pumping pyridine nucleotide transhydrogenase of Escherichia coli

• Published in: BBA - Bioenergetics 1998-06, Vol.1365 (1), p.98-104
• Main Author: Bragg, Philip D.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Binding Sites - genetics; Escherichia coli - enzymology; Escherichia coli - genetics; Inhibition site; Molecular Sequence Data; Mutagenesis, Site-Directed; NAD - metabolism; NADP - metabolism; NADP Transhydrogenases - chemistry; NADP Transhydrogenases - genetics; Protein Structure, Secondary; Proton pathway; Proton pumping; Pyridine nucleotide-binding site; Site-directed mutagenesis; Transhydrogenase
• ISSN: 0005-2728; 0006-3002; 1879-2650
• DOI: 10.1016/S0005-2728(98)00049-8

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 (α, β) organized as an α 2 β 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 α and β 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 α 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 β subunit. Site-directed matagenesis of the transmembrane domain of the β 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 matagenesis and amino acid deletions have shown that the six carboxy terminal residues of the α subunit and the two carboxy terminal residues of the β subunit are necessary for correct assembly of the transhydrogenase in the cytoplasmic membrane.