PMID:7592843
| Citation |
Lewendon, A, Ellis, J and Shaw, WV (1995) Structural and mechanistic studies of galactoside acetyltransferase, the Escherichia coli LacA gene product. J. Biol. Chem. 270:26326-31 |
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| Abstract |
Escherichia coli galactoside acetyltransferase (GAT) is a member of a large family of acetyltransferases that O-acetylate dissimilar substrates but share limited sequence homology. Steady-state kinetic analysis of over-expressed GAT demonstrated that it accepted a range of substrates, including glucosides and lactosides which were acetylated at rates comparable to galactosides. GAT was shown to be a trimeric acetyltransferase by cross-linking with dimethyl suberimidate. Fluorometric analysis of coenzyme A binding showed that there is a fluorescence quench associated with acetyl-CoA binding whereas CoA has no effect. This difference was exploited to measure dissociation rates for both CoA and acetyl-CoA by stopped-flow fluorometry. The rate of dissociation of CoA (2500 s-1) is at least 170-fold faster than kcat for any substrate tested. The fluorescence response to acetyl-CoA binding is entirely due to Trp-139 since replacement by phenylalanine completely abolished the fluorescence quench. Treatment of GAT by [14C]iodoacetamide resulted in complete inactivation of the enzyme and the incorporation of label into histidyl and cysteinyl residues to approximately equal extents. Following replacement of His-115 by alanine, label was incorporated solely into cysteinyl residues. Furthermore, the substitution results in an 1800-fold decrease in kcat suggesting that His-115 has an important catalytic role in GAT. |
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| Keywords |
Acetyl Coenzyme A/metabolism; Acetyltransferases/chemistry; Acetyltransferases/genetics; Acetyltransferases/metabolism; Base Sequence; Binding Sites; Cross-Linking Reagents/pharmacology; DNA Primers; Dimethyl Suberimidate/pharmacology; Escherichia coli/enzymology; Escherichia coli/genetics; Genes, Bacterial; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Spectrometry, Fluorescence; Substrate Specificity |
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