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 substra tes but share limited sequence homology. Steady-state kinetic analysis of overexpressed GAT demonstrated that it accepted a range of substra tes, including glucosides and lactosides which were acetylated at rate s comparable to galactosides. GAT was shown to be a trimeric acetyltra nsferase by cross-linking with dimethyl suberimidate. Fluorometric ana lysis of coenzyme A binding showed that there is a fluorescence quench associated with acetyl-CoA binding whereas CoA has no effect. This di fference was exploited to measure dissociation rates for both CoA and acetyl-CoA by stopped-flow fluorometry. The rate of dissociation of Co A (2500 s(-1)) is at least 170-fold faster than k(cat) for any substra te tested. The fluorescence response to acetyl-CoA binding is entirely due to Trp-139 since replacement by phenylalanine completely abolishe d the fluorescence quench. Treatment of GAT by [C-14]iodoacetamide res ulted in complete inactivation of the enzyme and the incorporation of label into histidyl and cysteinyl residues to approximately equal exte nts. Following replacement of His-115 by alanine, label was incorporat ed solely into cysteinyl residues. Furthermore, the substitution resul ts in an 1800-fold decrease in k(cat) suggesting that His-115 has an i mportant catalytic role in GAT.