Structural and mechanistic studies of galactoside acetyltransferase, the Escherichia coli LacA gene product.
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.
Lewendon
A
A
Department of Biochemistry, University of Leicester, United Kingdom.
Ellis
J
J
Shaw
W V
WV
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology; D017931:DNA Primers; D004120:Dimethyl Suberimidate / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D017931:DNA Primers; D011994:Recombinant Proteins; D004120:Dimethyl Suberimidate; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C022770:galactoside acetyltransferase