The presence of an aqueous cavity in the proton-pumping pathway of the pyridine nucleotide transhydrogenase of Escherichia coli is suggested by the reaction of the enzyme with sulfhydryl inhibitors.
The pyridine nucleotide transhydrogenase of Escherichia coli carries out transmembrane proton translocation coupled to transfer of a hydride ion equivalent between NAD(+) and NADP(+). The membrane domain (domain II) of the enzyme is composed of 13 transmembrane helices. Previous studies (N. A. Glavas et al., Biochemistry 34, 7694-7702, 1995) have suggested that betaHis91 in transmembrane helix 9 is involved in the translocation pathway of protons across the membrane. In this study we have replaced amino acid residues on the same face of helix 9 as betaHis91 by single cysteine residues. We then examined the effect of the sulfhydryl inhibitors N-ethylmaleimide (NEM) and p-chloromercuriphenylsulfonate (pCMPS) on enzyme activity and, in the case of [(14)C]NEM, as an enzyme label. The pattern of enzyme inhibition and labelling is consistent with the presence of an aqueous cavity through domain II from the cytosolic surface to the region of betaHis91. Residue betaAsn222 in helix 13, which appears also to be involved in the proton pathway across domain II, may interface with this aqueous cavity. A further series of mutants of betaGlu124 on helix 10 confirms the proposal (P. D. Bragg and C. Hou, Arch. Biochem. Biophys. 363, 182-190, 1999) that this residue is involved in passive permeation of protons across domain II.
Bragg
P D
PD
Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3, Canada. pbragg@interchange.ubc.ca
Hou
C
C
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011522:Protons; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013997:Time Factors; D014867:Water / Q000378:metabolism*
D004791:Enzyme Inhibitors; D011522:Protons; D013439:Sulfhydryl Reagents; D014867:Water; D002731:4-Chloromercuribenzenesulfonate; D009250:NADP Transhydrogenases; D003545:Cysteine; D005033:Ethylmaleimide