Acidic Residues Necessary for Pyrophosphate-energized Pumping and Inhibition of the Vacuolar H+-pyrophosphatase byN,N′-Dicyclohexylcarbodiimide*
Acidic Residues Necessary for Pyrophosphate-energized Pumping and Inhibition of the Vacuolar H+-pyrophosphatase byN,N′-Dicyclohexylcarbodiimide*
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DOI：
10.1074/jbc.272.35.22340
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发表时间：
1997-08
期刊：
影响因子：
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通讯作者：
R. Zhen;Eugene J. Kim;P. A. Rea
中科院分区：
文献类型：
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作者：
R. Zhen;Eugene J. Kim;P. A. Rea
根据两个结构预测程序TopPred II和MEMSAT对4个已发表的序列进行分析得到的液泡H+焦磷酸酶(V-PPase；EC 3.6.1.1)的拓扑模型，确定了8个位于跨膜α-螺旋附近或内部的酸性氨基酸残基。对拟南芥V-PPase编码基因中指定这些氨基酸的密码子进行了单突变，以研究它们在焦磷酸水解酶和依赖于焦磷酸的H+转运中的作用，以及V-PPase的Glu229227245和N‘-二环己基碳二亚胺结合的跨膜α-螺旋之间的相似性的功能意义。34、375-378)。突变酶在酿酒酵母中异源表达后，鉴定出三个功能类。I类(E119Q、E229Q、D573N、E667Q和E751Q)突变体表现出与野生型相似的PPI水解性和H+转运活性以及DCcd敏感性。获得的1个II类突变株(E427Q)对H+的转运优先于PPI的水解，但对DCD保持敏感。III类(E305Q和D504N)突变体几乎完全取消了PPI的水解和H+的转位，残存的活性降低了对DCD的敏感性。在所有突变体中，V-PPase插入膜中的量都没有减少，膜对H+的本底电导也没有明显的增加。与野生型相比，E427Q突变体的去偶联特性和突变体H+泵的增强，再加上E427Q和E427D突变体保留了DCCD的抑制能力，暗示了Glu427在V-PPase对DCCD不敏感的H+转运中的作用。E229Q突变体中PPI水解活性和H+转运活性的比例降低以及野生型DCD敏感性的保持驳斥了Glu229是其共价修饰的残基导致依赖于PPI的H+转运被取消的观点。相反，E305Q和D504N突变体的剩余活性对DCD抑制的敏感性降低，而E305D或D504E突变体则不是，这与这些位置的酸性残基参与了DCCD的抑制结合是一致的。关于Glu427可能参与PPI水解酶与跨膜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，本文对结果进行了讨论。
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 α-helices were identified. The codons specifying these amino acids in the cDNA encoding the V-PPase from Arabidopsis thalianawere 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 theN,N′-dicyclohexylcarbodiimide (DCCD)-binding transmembrane α-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.