Su Ping, Scheiner-Bobis Georgios
Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen, Frankfurter Strasse 100, D-35392 Giessen, Germany.
Biochemistry. 2004 Apr 27;43(16):4731-40. doi: 10.1021/bi049884f.
P-type ATPases such as the sodium pump appear to be members of a superfamily of hydrolases structurally typified by the L-2-haloacid dehalogenases. In the dehalogenase L-DEX-ps, Lys151 serves to stabilize the excess negative charge in the substrate/reaction intermediates and Asp180 coordinates a water molecule that is directly involved in ester intermediate hydrolysis. To investigate the importance of the corresponding Lys691 and Asp714 of the sodium pump alpha subunit, sodium pump mutants were expressed in yeast and analyzed for their properties. Lys691Ala, Lys691Asp, Asp714Ala, and Asp714Arg mutants were inactive, not only with respect to ATPase activity but also to interaction with the highly sodium pump-specific inhibitors ouabain or palytoxin (PTX). In contrast, conservative mutants Lys691Arg and Asp714Glu retained some of the partial activities of the wild-type enzyme, although they completely failed to display any ATPase activity. Yeast cells expressing Lys691Arg and Asp714Glu mutants are sensitive to the sodium pump-specific inhibitor PTX and lose intracellular K+. Their sensitivity to PTX, with EC50 values of 118 +/- 24 and 76.5 +/- 3.6 nM, respectively, was clearly reduced by almost 7- or 4-fold below that of the native sodium pump (17.8 +/- 2.7 nM). Ouabain was recognized under these conditions with low affinity by the mutants and inhibited the PTX-induced K+ efflux from the yeast cells. The EC50 for the ouabain effect was 183 +/- 20 microM for Lys691Arg and 2.3 +/- 0.08 mM for the Asp714Glu mutant. The corresponding value obtained with cells expressing the native sodium pump was 69 +/- 18 microM. In the presence of Pi and Mg2+, none of the mutant sodium pumps were able to bind ouabain. When Mg2+ was omitted, however, both Lys691Asp and Asp714Glu mutants displayed ouabain binding that was reduced by Mg2+ with an EC50 of 0.76 +/- 0.11 and 2.3 +/- 0.2 mM, respectively. In the absence of Mg2+, ouabain binding was also reduced by K+. The EC50 values were 1.33 +/- 0.23 mM for the wild-type enzyme, 0.93 +/- 0.2 mM for the Lys691Arg mutant, and 1.02 +/- 0.24 mM for the Asp714Glu enzyme. None of the neutral or nonconservative mutants displayed any ouabain-sensitive ATPase activity. Ouabain-sensitive phosphatase activity, however, was present in membranes containing either the wild-type (1105 +/- 100 micromol of p-nitrophenol phosphate hydrolyzed min(-1) mg of protein(-1)) or the Asp714Glu mutant (575 +/- 75 micromol min(-1) mg(-1)) sodium pump. Some phosphatase activity was also associated with the Lys691Arg mutant (195 +/- 63 micromol min(-1) mg(-1)). The results are consistent with Lys691 and Asp714 being essential for the phosphorylation/dephosphorylation process that allows the sodium pump to accomplish the catalytic cycle.
诸如钠泵之类的P型ATP酶似乎是水解酶超家族的成员，其结构以L-2-卤代酸脱卤酶为典型代表。在脱卤酶L-DEX-ps中，赖氨酸151用于稳定底物/反应中间体中的过量负电荷，而天冬氨酸180配位一个直接参与酯中间体水解的水分子。为了研究钠泵α亚基相应的赖氨酸691和天冬氨酸714的重要性，在酵母中表达钠泵突变体并分析其特性。赖氨酸691丙氨酸、赖氨酸691天冬氨酸、天冬氨酸714丙氨酸和天冬氨酸714精氨酸突变体均无活性，不仅在ATP酶活性方面，而且在与高度特异性的钠泵抑制剂哇巴因或岩沙海葵毒素（PTX）的相互作用方面。相反，保守突变体赖氨酸691精氨酸和天冬氨酸714谷氨酸保留了野生型酶的一些部分活性，尽管它们完全没有显示出任何ATP酶活性。表达赖氨酸691精氨酸和天冬氨酸714谷氨酸突变体的酵母细胞对钠泵特异性抑制剂PTX敏感并失去细胞内钾离子。它们对PTX的敏感性，其半数有效浓度（EC50）值分别为118±24和76.5±3.6 nM，明显比天然钠泵（17.8±2.7 nM）降低了近7倍或4倍。在这些条件下，突变体对哇巴因的亲和力较低，并抑制了PTX诱导的酵母细胞钾离子外流。赖氨酸691精氨酸突变体对哇巴因作用的EC50为183±20μM，天冬氨酸714谷氨酸突变体为2.3±0.08 mM。用表达天然钠泵的细胞获得的相应值为69±18μM。在有磷酸根离子（Pi）和镁离子（Mg2+）存在的情况下，没有一个突变型钠泵能够结合哇巴因。然而，当省略镁离子时，赖氨酸691天冬氨酸和天冬氨酸714谷氨酸突变体均显示出哇巴因结合，其结合被镁离子降低，EC50分别为0.76±0.11和2.3±0.2 mM。在没有镁离子的情况下，哇巴因结合也被钾离子降低。野生型酶的EC50值为1.33±0.23 mM，赖氨酸691精氨酸突变体为0.93±0.2 mM，天冬氨酸714谷氨酸酶为1.02±0.24 mM。没有一个中性或非保守突变体显示出任何对哇巴因敏感的ATP酶活性。然而，在含有野生型（每分钟水解对硝基苯磷酸1105±100微摩尔每毫克蛋白质）或天冬氨酸714谷氨酸突变体（575±75微摩尔每分钟每毫克）钠泵的膜中存在对哇巴因敏感的磷酸酶活性。一些磷酸酶活性也与赖氨酸691精氨酸突变体相关（195±63微摩尔每分钟每毫克）。结果与赖氨酸691和天冬氨酸714对于使钠泵完成催化循环的磷酸化/去磷酸化过程至关重要这一观点一致。
