识别的组氨酸的3-hydroxy-3-methylglutaryl-coenzyme还原酶催化地重要。
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王达BG, Y,罗德威尔大众
识别的组氨酸的3-hydroxy-3-methylglutaryl-coenzyme还原酶催化地重要。
J生物化学杂志。1992年7月25日,267 (21):15064 - 70。
- PubMed ID
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1634543 (在PubMed]
- 文摘
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我们识别假单胞菌的His381 mevalonii 3-hydroxy-3-methylglutaryl-coenzyme(β)还原酶催化残的基本功能。催化领域的20β-还原酶包含单个守恒的组氨酸(p . mevalonii His381酶)。焦碳酸二乙酯灭活酶p . mevalonii,羟胺部分恢复活动。我们改变了His381丙氨酸,赖氨酸,天冬酰胺,谷氨酰胺。突变体蛋白过表达,纯化同质性,特征。焦碳酸二乙酯His381突变酶不灭活。所有四个突变酶表现出野生型晶体形态学和色谱仪支持像野生型酶底物的亲和力。突变体酶催化活性较低(0.06 Vmax野生型酶的-0.5%),但公里价值近似的野生型酶。H381N,野生型和突变体酶酶H381A H381Q,在pH值8.1公里值分别为0.45,0.27,3.7,和0.71毫米((R, S)甲羟戊酸);0.05,0.03,0.20,和0.11毫米(辅酶A); 0.22, 0.14, 0.81, and 0.62 mM [NAD+]. Km values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM [(R,S)-mevalonate]; 0.24 and 0.50 mM [coenzyme A]; 0.15 and 1.23 mM [NAD+]. Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK1 and by 0.5-1.3 pH units for pK2). For mutant enzyme H381K, the pK1 of 10.2 is consistent with lysine acting as a general base at high pH. His381 of P. mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.