Mueller D M
Department of Biological Chemistry and Structure, University of Health Sciences, Chicago Medical School, Illinois 60064.
J Biol Chem. 1988 Apr 25;263(12):5634-9.
The mitochondrial ATPase is rapidly inactivated by the arginine selective reagent phenylglyoxal. Recently, the purported major reacting residue has been reported for the chloroplast enzyme (Viale, A. M., and Vallejos, R. H. (1985) J. Biol. Chem. 260, 4958-4962) corresponding to Arg-328 in the beta-subunit of the yeast Saccharomyces cerevisiae mitochondrial ATPase, a highly conserved residue in the ATPase. This arginine residue was concluded to be in the active site of the ATPase and possibly involved in the binding of nucleotides. To test this hypothesis, site-directed mutagenesis of the yeast enzyme has been used to replace Arg-328 with alanine and lysine. The modified genes were transformed into a yeast strain, DMY111, which contained a null mutation in the gene coding for the beta-subunit of the ATPase. Both of the substitutions were functional in vivo as demonstrated by the ability of yeast transformants to grow on a nonfermentable carbon source. The water soluble F1-ATPase with Ala-328 and Lys-328 were extremely unstable, but could be stabilized with glycerol. The rate of enzymatic decay followed first order kinetics with half-lives of 1.1 and 4.0 min for the mutants with Ala-328 and Lys-328 in 10% and 5% glycerol, respectively, while the wild type enzyme was stable even in the absence of glycerol. Kinetic analysis of both ATPase and GTPase has been determined. The wild type enzyme had two observable apparent Km and Vmax values for ATPase which were 0.056 mM-1 and 67 units/min/mg and 0.140 mM-1 and 100 units/min/mg. The mutant enzyme containing Lys-328 showed similar kinetic values of 0.066 mM-1 and 23 units/min/mg and 0.300 mM-1 and 43 units/min/mg. The mutant enzyme containing Ala-328, however, only demonstrated a single site with values of 0.121 mM-1 and 45 units/min/mg. In contrast to ATPase activity, kinetic values for GTPase were nearly identical for the wild type and mutant enzymes. Opposite to predicted results, the mutant enzymes were more sensitive to the reagent phenylglyoxal. These results indicate that Arg-328 is important for protein stability, but not involved in catalysis.
线粒体ATP酶可被精氨酸选择性试剂苯乙二醛迅速灭活。最近，已报道了叶绿体酶的主要反应残基（维亚莱，A.M.，和巴列霍斯，R.H.（1985年）《生物化学杂志》260，4958 - 4962），它对应于酿酒酵母线粒体ATP酶β亚基中的精氨酸-328，这是ATP酶中一个高度保守的残基。据推测，这个精氨酸残基位于ATP酶的活性位点，可能参与核苷酸的结合。为了验证这一假设，已利用酵母酶的定点诱变将精氨酸-328替换为丙氨酸和赖氨酸。修饰后的基因被转化到酵母菌株DMY111中，该菌株在编码ATP酶β亚基的基因中存在无效突变。正如酵母转化体在不可发酵碳源上生长的能力所证明的那样，这两种替换在体内都是有功能的。含有丙氨酸-328和赖氨酸-328的水溶性F1 - ATP酶极其不稳定，但可用甘油使其稳定。酶的衰变速率遵循一级动力学，在10%甘油中，含有丙氨酸-328的突变体半衰期为1.1分钟，含有赖氨酸-328的突变体半衰期为4.0分钟；而野生型酶即使在没有甘油的情况下也是稳定的。已对ATP酶和GTP酶进行了动力学分析。野生型酶对ATP酶有两个可观察到的表观Km和Vmax值，分别为0.056 mM-1和67单位/分钟/毫克以及0.140 mM-1和100单位/分钟/毫克。含有赖氨酸-328的突变酶显示出类似的动力学值，分别为0.066 mM-1和23单位/分钟/毫克以及0.300 mM-1和43单位/分钟/毫克。然而，含有丙氨酸-328的突变酶仅显示出一个位点，值为0.121 mM-1和45单位/分钟/毫克。与ATP酶活性相反，野生型和突变型酶的GTP酶动力学值几乎相同。与预测结果相反，突变酶对试剂苯乙二醛更敏感。这些结果表明，精氨酸-328对蛋白质稳定性很重要，但不参与催化作用。
