Meng M, Chuang D T
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.
Biochemistry. 1994 Nov 1;33(43):12879-85. doi: 10.1021/bi00209a020.
The catalytic domain of dihydrolipoamide transacylase (E2c) of bovine branched-chain alpha-keto acid dehydrogenase complex (BCKAD) was overexpressed in Escherichia coli. The E2c catalyzes a reversible acyl transfer reaction between acyl-CoA and dihydrolipoamide, which also occurs spontaneously with a much slower rate. The benzene extracts of both the enzyme-catalyzed and the spontaneous reactions mixture have identical ultraviolet absorbance spectra with a maximum at 233-234 nm, which is characteristic of S-acyldihydrolipoamide. The spontaneous reaction rate of various acyl-CoA is in the order of acetoacetyl-CoA > acetyl-CoA > isobutyryl-CoA > isovaleryl-CoA. In other words, the spontaneous acyl transfer is faster when the substituent (R) of acyl-CoA (R-CO-S-CoA) is a more electron-withdrawing group. This result indicates that a negative charge occurs in the substrate during the acyl transfer process. The function of the active-site histidine (His391) and serine (Ser338) of bovine E2c was analyzed by site-directed mutagenesis. Substitution of His391 or Ser338 with alanine caused drastic decreases in catalytic efficiencies by 3-4 orders of magnitude. The residual activity of H391A increased as the pH of the reaction buffer was elevated. These data support the base-catalyzed mechanism inferred from that of chloramphenicol acetyltransferase (CAT). In this reaction, the active-site histidine acts as a general base, and the active-site serine provides a hydrogen bond to the putative negatively charged tetrahedral transition state. Moreover, when Ala348 was changed to valine, the catalytic efficiency for isovaleryl-CoA decreased about 10-fold, and that for acetyl-CoA increased about 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
牛支链α-酮酸脱氢酶复合体（BCKAD）的二氢硫辛酰胺转乙酰酶（E2c）催化结构域在大肠杆菌中过表达。E2c催化酰基辅酶A和二氢硫辛酰胺之间的可逆酰基转移反应，该反应也能自发发生，但速率要慢得多。酶催化反应混合物和自发反应混合物的苯提取物具有相同的紫外吸收光谱，最大吸收峰在233 - 234 nm处，这是S-酰基二氢硫辛酰胺的特征。各种酰基辅酶A的自发反应速率顺序为：乙酰乙酰辅酶A＞乙酰辅酶A＞异丁酰辅酶A＞异戊酰辅酶A。换句话说，当酰基辅酶A（R-CO-S-CoA）的取代基（R）是更强的吸电子基团时，自发酰基转移更快。这一结果表明在酰基转移过程中底物会产生负电荷。通过定点诱变分析了牛E2c活性位点组氨酸（His391）和丝氨酸（Ser338）的功能。用丙氨酸取代His391或Ser338会导致催化效率急剧下降达3 - 4个数量级。H391A的残余活性随着反应缓冲液pH值的升高而增加。这些数据支持了从氯霉素乙酰转移酶（CAT）推断出的碱催化机制。在该反应中，活性位点组氨酸作为通用碱，活性位点丝氨酸为假定的带负电荷的四面体过渡态提供氢键。此外，当Ala348变为缬氨酸时，对异戊酰辅酶A的催化效率降低约10倍，对乙酰辅酶A的催化效率增加约3倍。（摘要截短至250字）
