SITE-DIRECTED MUTAGENESIS AND FUNCTIONAL-ANALYSIS OF THE ACTIVE-SITE RESIDUES OF THE E2 COMPONENT OF BOVINE BRANCHED-CHAIN ALPHA-KETO ACID DEHYDROGENASE COMPLEX

The catalytic domain of dihydrolipoamide transacylase (E2c) of bovine branched-chain a-keto acid dehydrogenase complex (BCKAD) was overexpre ssed in Escherichia coli. The E2c catalyzes a reversible acyl transfer reaction between acyl-CoA and dihydrolipoamide, which also occurs spo ntaneously with a much slower rate. The benzene extracts of both the e nzyme-catalyzed and the spontaneous reactions mixture have identical u ltraviolet absorbance spectra with a maximum at 233-234 nm, which is c haracteristic 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 acy l 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 ne gative charge occurs in the substrate during the acyl transfer process . The function of the active-site histidine (His391) and serine (Ser33 8) of bovine E2c was analyzed by site-directed mutagenesis. Substituti on of His391 or Ser338 with alanine caused drastic decreases in cataly tic efficiencies by 3-4 orders of magnitude. The residual activity of H391A increased as the pH of the reaction buffer was elevated. These d ata support the base-catalyzed mechanism inferred from that of chloram phenicol acetyltransferase (CAT). In this reaction, the active-site hi stidine acts as a general base, and the active-site serine provides a hydrogen bond to the putative negatively charged tetrahedral transitio n state, Moreover, when Ala348 was changed to valine, the catalytic ef ficiency for isovaleryl-CoA decreased about 10-fold, and that for acet yl-CoA increased about 3-fold. Ala348 presumably contacts the isobutyl group of isovaleryl-CoA in the acyl transfer reaction. Our results in dicate that this residue plays a key role in the substrate preference of bovine E2c.