酿酒酵母支链α-酮酸脱氢酶复合体的纯化与特性分析

Purification and characterization of the branched chain alpha-ketoacid dehydrogenase complex from Saccharomyces cerevisiae.

作者信息

Sinclair D A, Dawes I W, Dickinson J R

机构信息

School of Biochemistry and Molecular Genetics, University of New South Wales, Kensington, Australia.

出版信息

Biochem Mol Biol Int. 1993 Dec;31(5):911-22.

PMID:8136709

Abstract

Branched chain alpha-ketoacid dehydrogenase complex was purified from Saccharomyces cerevisiae by polyethylene glycol fractionation and chromatography on Sephacryl S-200, DEAE-cellulose and Sepharose CL-2B. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicated the enzyme contained subunits of M(r) = 57,000, 52,000, 47,000 and 38,000. The specific activity of the purified enzyme was 0.82 mumol NADH/min/mg protein at 30 degrees C with 16 mM alpha-ketoisovalerate as substrate. The apparent Km values for alpha-ketoisovalerate, alpha-ketoisocaproate and alpha-keto-beta-methylvalerate were 21, 22, and 20 mM, respectively. The preparation was also able to oxidize the intermediates of threonine and methionine metabolism, alpha-keto-gamma-methiolbutyrate and alpha-ketobutyrate, with Km values of 13 and 8 mM, respectively.

摘要

通过聚乙二醇分级分离以及在Sephacryl S - 200、DEAE - 纤维素和琼脂糖CL - 2B上进行层析，从酿酒酵母中纯化出支链α - 酮酸脱氢酶复合体。在十二烷基硫酸钠 - 聚丙烯酰胺凝胶上进行电泳表明，该酶含有分子量分别为57,000、52,000、47,000和38,000的亚基。在30℃下，以16 mMα - 酮异戊酸为底物时，纯化酶的比活性为0.82 μmol NADH/分钟/毫克蛋白。α - 酮异戊酸、α - 酮异己酸和α - 酮 - β - 甲基戊酸的表观Km值分别为21、22和20 mM。该制剂还能够氧化苏氨酸和蛋氨酸代谢的中间体α - 酮 - γ - 甲硫基丁酸和α - 酮丁酸，其Km值分别为13和8 mM。