Griffin T A, Chuang D T
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1990 Aug 5;265(22):13174-80.
Genetically altered transacylase (E2b) proteins of the bovine branched-chain alpha-keto acid dehydrogenase complex were overexpressed in Escherichia coli and characterized. Deletion by PstI or Bal31 digestion of the amino-terminal region of the inner-core domain (residues 175-421) beyond residue 209 resulted in a complete loss of transacylase activity. The enzyme assay was carried out using [1-14C]isovaleryl-CoA and exogenous dihydrolipoamide as substrates. The removal of 4 residues (Thr-Ile-Pro-Ile) (residues 175-178) from the amino terminus of the inner-core domain significantly reduced the level of transacylase activity. The results establish that the segment between residues 175 and 209 is an integral part of the active site of E2b. The residue His-391 in the recombinant inner-core domain (E2b delta 167) was changed to Asn or Gln by site-directed mutagenesis. The wild-type and the two mutant inner-core domains were assembled into 24-mers as determined by gel filtration. However, both Asn and Gln mutations were accompanied by a complete loss of the enzymatic activity. Titration of the natural branched-chain alpha-keto dehydrogenase complex from pH 8 to 6 sharply reduced transacylase activity. The above data support the hypothesis that a conserved histidine residue in E2 acts as a general base for the transacylation reaction by analogy with E. coli chloramphenicol acetyltransferases.
牛支链α-酮酸脱氢酶复合体的基因工程改造转酰酶（E2b）蛋白在大肠杆菌中过表达并进行了特性分析。用PstI或Bal31消化内核结构域（残基175 - 421）中209位残基以外的氨基末端区域，导致转酰酶活性完全丧失。酶活性测定使用[1-¹⁴C]异戊酰辅酶A和外源性二氢硫辛酰胺作为底物。从内核结构域的氨基末端去除4个残基（苏氨酸-异亮氨酸-脯氨酸-异亮氨酸）（残基175 - 178）显著降低了转酰酶活性水平。结果表明，175位和209位残基之间的片段是E2b活性位点的一个组成部分。通过定点诱变将重组内核结构域（E2b δ167）中的组氨酸-391残基变为天冬酰胺或谷氨酰胺。通过凝胶过滤测定，野生型和两个突变内核结构域组装成了24聚体。然而，天冬酰胺和谷氨酰胺突变均伴随着酶活性的完全丧失。将天然支链α-酮脱氢酶复合体从pH 8滴定到pH 6会急剧降低转酰酶活性。上述数据支持这样的假说：E2中一个保守的组氨酸残基类似于大肠杆菌氯霉素乙酰转移酶，作为转酰化反应的通用碱。
