Structural and mechanistic studies of galactoside acetyltransferase, the Escherichia coli LacA gene product

Abstract

Escherichia coil 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 heat 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 alanin, label was incorporated solely into cysteinyl residues. Furthermore, the substitution results in an 1800-fold decrease in k(cat) suggesting that His- IlS has an important catalytic role in GAT.