The catalytic domain of dihydrolipoamide transacylase (E2c) of bovine branched-chain α-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. Ala348 presumably contacts the isobutyl group of isovaleryl-CoA in the acyl transfer reaction. Our results indicate that this residue plays a key role in the substrate preference of bovine E2c.
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