Mutation of conserved polar residues in the transmembrane domain of the proton-pumping pyridine nucleotide transhydrogenase of Escherichia coli

被引:20
作者
Bragg, PD [1 ]
Hou, C [1 ]
机构
[1] Univ British Columbia, Dept Biochem & Mol Biol, Vancouver, BC V6T 1Z3, Canada
基金
英国医学研究理事会;
关键词
transhydrogenase; proton pumping; membrane mutants; pyridine nucleotide transhydrogenase; proton pathway; membrane domain;
D O I
10.1006/abbi.1998.1062
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The pyridine nucleotide transhydrogenase carries out transmembrane proton translocation coupled to transfer of a hydride ion equivalent between NAD(+) and NADP(+). Previous workers (E. Holmberg et at Biochemistry 33, 7691-7700, 1994; N. A. Glavas et al. Biochemistry 34, 7694-7702, 1995) had examined the role in proton translocation of conserved charged residues in the transmembrane domain. This study was extended to examine the role of conserved polar residues of the transmembrane domain. Site-directed mutagenesis of these residues did not produce major effects on hydride transfer or proton translocation activities except in the case of beta Asn222. Most mutants of this residue were drastically impaired in these activities. Three phenotypes were recognized. In beta N222C both activities were impaired maximally by 70%. The retention of proton translocation indicated that beta Asn222 was not directly involved in proton translocation, In beta N222H both activities were drastically reduced. Binding of NADP(+) but not of NADPH was impaired. In beta N222R, by contrast, NADP(+) remained tightly bound to the mutant transhydrogenase. It is concluded that beta Asn222, located in a transmembrane alpha-helix, is part of the conformational pathway by which NADP(H) binding, which occurs outside of the transmembrane domain, is coupled to proton translocation. Some nonconserved or semiconserved polar residues of the transmembrane domain were also examined by site-directed mutagenesis. Interaction of beta Glu124 with the proton translocation pathway is proposed. (C) 1999 Academic Press.
引用
收藏
页码:182 / 190
页数:9
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