识别的主要催化地重要的酸性渣3-hydroxy-3-methylglutaryl辅酶A还原酶。
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王Y·达尼BG,罗德威尔大众
识别的主要催化地重要的酸性渣3-hydroxy-3-methylglutaryl辅酶A还原酶。
生物化学杂志。1990年12月15日;265 (35):21634 - 41。
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2123872 (在PubMed]
- 文摘
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动力学分析3-hydroxy-3-methylglutaryl辅酶A还原酶(β)有牵连谷氨酸或天冬氨酸残基(i)形成mevaldate thiohemiacetal通过质子转移的羰基氧mevaldate和(2)增强电离产生的酶的CoASH羧酸盐阴离子,促进攻击弱点-羰基碳的mevaldate(维罗索,D。克莱兰德,W W。波特,j . w .(1981)生物化学81 887 - 894)。尽管这种酸性渣的身份和它的位置是已知的,催化域11测序β-还原酶只包含3守恒的酸性残基。假单胞菌β-还原酶的mevalonii,这些残留物Glu52, Glu83, Asp183。识别功能的酸性渣催化,我们生成的突变体改变这些残留物。突变蛋白的表达、纯化和表征。突变蚀变残留Glu52或Asp183 p mevaloniiβ-还原酶产生酶显著,但在某些情况下减少,活动(Vmax = 100% Asp183——阿拉巴马州,Asp183——Asn, 65%和15% Glu52——Gln的野生型活动,分别)。尽管突变酶的活性Glu52——Gln和Asp183——阿拉巴马州无法在标准试验条件下,他们的Km值基质4 - 300倍高于野生型酶。公里值为野生型酶和突变酶Glu52——Gln和Asp183——阿拉巴马州,分别为:0.41,73,和120毫米(R, S)甲羟戊酸);0.080、4.4和2.0毫米(辅酶); and 0.26, 4.4, and 1.0 mM (NAD+). By these criteria, neither Glu52 nor Asp183 is the acidic catalytic residue although each may function in substrate recognition. During chromatography on coenzyme A agarose or HMG-CoA agarose, mutant enzymes Asp183----Asn and Glu83----Gln behaved like wild-type enzyme. By contrast, and in support of a role for these residues in substrate recognition, mutant enzymes Glu52----Gln and Asp183----Ala exhibited impaired ability to bind to either support. Despite displaying Km values for substrates and chromatographic behavior on substrate affinity supports comparable to wild-type enzyme, only mutant enzyme Glu83----Gln was essentially inactive under all conditions studied (Vmax = 0.2% that of wild-type enzyme). Glutamate residue 83 of P. mevalonii HMG-CoA reductase, and consequently the glutamate of the consensus Pro-Met-Ala-Thr-Thr-Glu-Gly-Cys-Leu-Val-Ala motif of the catalytic domains of eukaryotic HMG-CoA reductases, is judged to be the acidic residue functional in catalysis.