PROBING THE ROLE OF CYSTEINE RESIDUES IN GLUCOSAMINE-1-PHOSPHATE ACETYLTRANSFERASE ACTIVITY OF THE BIFUNCTIONAL GLMU PROTEIN FROM ESCHERICHIA-COLI - SITE-DIRECTED MUTAGENESIS AND CHARACTERIZATION OF THE MUTANTENZYMES

The glucosamine-1-phosphate acetyltransferase activity but not the uri dyltransferase activity of the bifunctional GlmU enzyme from Escherich ia coil was lost when GlmU was stored in the absence of beta-mercaptoe thanol or incubated with thioI-specific reagents. The enzyme was prote cted from inactivation in the presence of its substrate acetyl coenzym e A (acetyl-CoA), suggesting the presence of an essential cysteine res idue in or near the active site of the acetyltransferase domain. To as certain the role of cysteines in the structure and function of the enz yme, site-directed mutagenesis was performed to change each of the fou r cysteines to alanine, and plasmids were constructed for high-level o verproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared una ffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutati ons, respectively. The K-m values for acetyl-CoA and GlcN-1-P of mutan t proteins were not modified, suggesting that none of the cysteines wa s involved in substrate binding. The uridyltransferase activities of w ildtype and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransfer ase activity and seemed to be located in or near the active site.