Zhen R G, Kim E J, Rea P A
Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018, USA.
J Biol Chem. 1997 Aug 29;272(35):22340-8. doi: 10.1074/jbc.272.35.22340.
On the basis of a revised topological model of the vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) derived from the analysis of four published sequences using two structure-predicting programs, TopPred II and MEMSAT, eight acidic amino acid residues located near or within transmembrane alpha-helices were identified. The codons specifying these amino acids in the cDNA encoding the V-PPase from Arabidopsis thaliana were singly mutated to examine their involvement in pyrophosphate (PPi) hydrolysis and PPi-dependent H+ translocation and the functional significance of the similarities between the sequences encompassing Glu229 (227-245) of the V-PPase and the N,N'-dicyclohexylcarbodiimide (DCCD)-binding transmembrane alpha-helix of the c-subunits of F-ATPases (Nyren, P., Sakai-Nore, Y. , and Strid, A. (1993) Plant Cell Physiol. 34, 375-378). Three functional classes were identified after heterologous expression of mutated enzyme in Saccharomyces cerevisiae. Class I (E119Q, E229Q, D573N, E667Q, and E751Q) mutants exhibited PPi hydrolytic and H+ translocation activities and DCCD sensitivities similar to wild type. The one class II mutant obtained (E427Q) was preferentially impaired for H+ translocation over PPi hydrolysis but retained sensitivity to DCCD. Class III (E305Q and D504N) mutants exhibited a near complete abolition of both PPi hydrolysis and H+ translocation and residual activities with decreased DCCD sensitivity. In none of the mutants was diminished insertion of the V-PPase into the membrane or an increase in the background conductance of the membrane to H+ evident. The decoupled character of E427Q mutants and the enhancement of H+ pumping in E427D mutants by comparison with wild type, in conjunction with the retention of DCCD inhibitability in both E427Q and E427D mutants, implicate a role for Glu427 in DCCD-insensitive H+ translocation by the V-PPase. The proportionate diminution of PPi hydrolytic and H+ translocation activity and conservation of wild type DCCD sensitivity in E229Q mutants refute the notion that Glu229 is the residue whose covalent modification by DCCD is responsible for the abolition of PPi-dependent H+ translocation. Instead, the diminished sensitivity of the residual activities of E305Q and D504N mutants, but not E305D or D504E mutants, to inhibition by DCCD is consistent with the involvement of acidic residues at these positions in inhibitory DCCD binding. The results are discussed with regard to the possible involvement of Glu427 in coupling PPi hydrolysis with transmembrane H+ translocation and earlier interpretations of the susceptibility of the V-PPase to inhibition by carbodiimides.
基于使用两种结构预测程序TopPred II和MEMSAT对四个已发表序列进行分析后得出的液泡H⁺ - 焦磷酸酶（V - PPase；EC 3.6.1.1）的修订拓扑模型，确定了位于跨膜α - 螺旋附近或内部的八个酸性氨基酸残基。对拟南芥V - PPase编码cDNA中指定这些氨基酸的密码子进行单突变，以研究它们在焦磷酸（PPi）水解和PPi依赖性H⁺转运中的作用，以及V - PPase中包含Glu229（227 - 245）的序列与F - ATP酶c亚基的N，N' - 二环己基碳二亚胺（DCCD）结合跨膜α - 螺旋之间相似性的功能意义（Nyren，P.，Sakai - Nore，Y.，和Strid，A.（1993）植物细胞生理学。34，375 - 378）。在酿酒酵母中对突变酶进行异源表达后，鉴定出了三个功能类别。I类（E119Q、E229Q、D573N、E667Q和E751Q）突变体表现出与野生型相似的PPi水解和H⁺转运活性以及DCCD敏感性。获得的一个II类突变体（E427Q）在H⁺转运方面比PPi水解更优先受损，但仍保留对DCCD的敏感性。III类（E305Q和D504N）突变体表现出PPi水解和H⁺转运几乎完全丧失，以及残留活性且DCCD敏感性降低。在任何突变体中，均未发现V - PPase插入膜的减少或膜对H⁺的背景电导率增加。与野生型相比，E427Q突变体的解偶联特性以及E427D突变体中H⁺泵浦的增强，再加上E427Q和E427D突变体中DCCD抑制性的保留，表明Glu427在V - PPase的DCCD不敏感H⁺转运中起作用。E229Q突变体中PPi水解和H⁺转运活性的成比例降低以及野生型DCCD敏感性的保留，驳斥了Glu229是其被DCCD共价修饰导致PPi依赖性H⁺转运丧失的残基这一观点。相反，E305Q和D504N突变体的残留活性对DCCD抑制的敏感性降低，但E305D或D５04E突变体则不然，这与这些位置的酸性残基参与抑制性DCCD结合一致。结合Glu427可能参与PPi水解与跨膜H⁺转运的偶联以及对V - PPase对碳二亚胺抑制敏感性的早期解释，对结果进行了讨论。
