Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
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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)
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PMID:Site-directed mutagenesis and functional analysis of the active-site residues of the E2 component of bovine branched-chain alpha-keto acid dehydrogenase complex. 794 94