The role of conserved histidine residues in the 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 translocation of protons across the cytoplasmic membrane. The role of histidine residues in catalysis was investigated by chemical modification with diethylpyrocarbonate and by site-directed mutagenesis. Diethylpyrocarbonate inhibited both hydride ion transfer and coupled proton translocation. Histidine residues were modified as shown spectroscopically and by the ability of hydroxylamine to cause reversal of inhibition. Complete inhibition of hydride ion transfer occurred following modification of 10 residues/enzyme molecule. Site-directed mutagenesis of single conserved histidine residues or the presence of substrates did not provide resistance to inhibition by diethylpyrocarbonate. It is concluded that diethylpyrocarbonate inhibition was a consequence of the structural changes brought about by modification of many histidine residues. With the exception of beta-subunit residue His91 (beta His91), in which mutation can result in specific loss of proton translocation activity [Glavas, N. A., Hou, C. & Bragg, P. D. (1995) Biochemistry 34, 7694-7702], site-directed mutation of the remaining conserved residues alpha His450, beta His161, beta His345 and beta His354 did not demonstrate a direct role for these residues in catalysis. Mutation of beta His161 had relatively little effect on the properties of the enzyme. By contrast, mutation of alpha His450, beta His345 and beta His354 caused major loss of enzyme activities which was probably due to alterations in the structure of the enzyme. These alterations were reflected in changes in the K(m) values for transhydrogenation.
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
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D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011522:Protons; D013379:Substrate Specificity; D014357:Trypsin
D004791:Enzyme Inhibitors; D011522:Protons; D009243:NAD; D006639:Histidine; D009250:NADP Transhydrogenases; D014357:Trypsin; D004047:Diethyl Pyrocarbonate