Jordan-Starck T C, Rodwell V W
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 1989 Oct 25;264(30):17913-8.
Pseudomonas mevalonii (formerly designated Pseudomonas sp. M (Beach, M. J., and Rodwell, V. W. (1989) J. Bacteriol. 171, 2994-3001; Gill, J. F., Jr., Beach, M.J., and Rodwell, V. W. (1985) J. Biol. Chem. 260, 9393-9398] 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.88), overexpressed in Escherichia coli (1), has been purified to electrophoretic homogeneity in 75% yield (final specific activity 48 mumols of NAD+ reduced per min/mg protein). The enzyme catalyzes its normal catabolic reaction (mevalonate + 2 NAD+ + CoASH----HMG-CoA + 2NADH + 2H+), and two half-reactions which involve mevaldehyde, the postulated intermediate in the aforementioned reactions and mevaldehyde + NADH + H+----mevalonate + NAD+). The rates of all four reactions and the Michaelis constants for all substrates were measured. Coenzyme A decreased the KM for mevaldehyde reduction 12-fold and stimulated VMAX 2-3 fold. CoASH thus may remain bound throughout the catalytic cycle. Dithiothreitol and analogs of CoASH were tested for their ability to reproduce the CoASH stimulation. Pantetheine, but not dithiothreitol, pantothenate, or desulfo-CoA mimicked CoASH stimulation. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) indicated two sulfhydryl groups per subunit. Both groups remained accessible to 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of mevalonate and/or NAD+ but only one group in the presence of HMG-CoA. N-Ethylmaleimide inhibited all the aforementioned reactions. HMG-CoA, but not mevalonate, afforded protection completely and irreversibly inactivated the enzyme. The reactive sulfhydryl group thus may not be a catalytic residue, but may be involved in a conformational change.
梅瓦洛尼假单胞菌（以前称为假单胞菌属M [比奇，M. J.，和罗德韦尔，V. W.（1989年）《细菌学杂志》171卷，2994 - 3001页；吉尔，J. F.，小，比奇，M. J.，和罗德韦尔，V. W.（1985年）《生物化学杂志》260卷，9393 - 9398页]）的3 - 羟基 - 3 - 甲基戊二酰辅酶A（HMG - CoA）还原酶（EC 1.1.1.88），在大肠杆菌中过表达（1），已以75%的产率纯化至电泳纯（最终比活性为每分钟每毫克蛋白质还原48微摩尔NAD⁺）。该酶催化其正常的分解代谢反应（甲羟戊酸 + 2NAD⁺ + 辅酶A→HMG - CoA + 2NADH + 2H⁺），以及两个半反应，其中涉及甲羟醛，上述反应中的假定中间体以及甲羟醛 + NADH + H⁺→甲羟戊酸 + NAD⁺）。测定了所有四个反应的速率以及所有底物的米氏常数。辅酶A使甲羟醛还原的KM降低了12倍，并使VMAX提高了2 - 3倍。因此，辅酶A在整个催化循环中可能保持结合状态。测试了二硫苏糖醇和辅酶A类似物再现辅酶A刺激作用的能力。泛硫乙胺，但不是二硫苏糖醇、泛酸盐或脱硫辅酶A，模拟了辅酶A的刺激作用。用5,5'-二硫代双（2 - 硝基苯甲酸）滴定表明每个亚基有两个巯基。在甲羟戊酸和/或NAD⁺存在下，这两个基团都可被5,5'-二硫代双（2 - 硝基苯甲酸）接近，但在HMG - CoA存在下只有一个基团可被接近。N - 乙基马来酰亚胺抑制所有上述反应。HMG - CoA，但不是甲羟戊酸，能提供完全保护并使酶不可逆失活。因此，反应性巯基可能不是催化残基，但可能参与构象变化。
