Bragg P D
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Biochim Biophys Acta. 1998 Jun 10;1365(1-2):98-104. 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 (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.
大肠杆菌的吡啶核苷酸转氢酶催化氢离子等价物在NAD⁺和NADP⁺之间的可逆转移，并与质子跨细胞质膜的转运相偶联。它由两个亚基（α、β）组成，组装成α₂β₂四聚体。这篇简短的综述描述了如何使用定点诱变来研究转氢酶的结构、机制和组装。该技术分别在α和β亚基中定位了NAD(H)和NADP(H)的结合位点。诱变表明，该酶的半胱氨酸残基对其功能并非必不可少，并且巯基特异性试剂对该酶的抑制作用必定是由于三维结构的扰动所致。已经确定了抑制剂N,N'-二环己基碳二亚胺和N-(1-芘基)马来酰亚胺的反应位点。选择性突变和半胱氨酸残基的插入，随后经邻菲罗啉铜诱导的二硫键交联，确定了全酶中α亚基之间的相互作用区域。通过测定选择性插入的半胱氨酸残基的可及性来确定β亚基跨膜螺旋的折叠模式。对β亚基跨膜结构域进行定点诱变，使得能够鉴定出作为转氢酶质子泵浦途径一部分的组氨酸、天冬氨酸和天冬酰胺残基。定点诱变和氨基酸缺失表明，α亚基的六个羧基末端残基和β亚基的两个羧基末端残基对于转氢酶在细胞质膜中的正确组装是必需的。
