Active-site mapping and site-specific mutagenesis of glycinamide ribonucleotide transformylase from Escherichia coli.
The affinity reagent N10-(bromoacetyl)-5,8-dideazafolate has previously been shown to inactivate glycinamide ribonucleotide transformylase (EC 2.1.2.2) from Escherichia coli in an active-site-directed manner with a 1:1 stoichiometry [Inglese et al. (1990) Biochemistry 29, 1436-1443]. After a series of mild proteolytic digestions, the dideazafolate label was localized to an active-site peptide attached by an ester linkage to the highly conserved residue Asp 144. Subsequent site-specific mutagenesis of Asp 144 to Asn 144 resulted in a catalytically inactive enzyme that retained the ability to bind substrates and inhibitors. The Asn 144 mutant could be further labeled with the affinity reagent in an active-site-directed stoichiometric fashion; however, the site of modification in this case was His 119. These results imply that Asp 144 may function as a general base within the catalytic center of the transformylase and is in close proximity to His 119 in the folded protein.
Department of Chemistry, Pennsylvania State University, University Park 16801.
J
J
Inglese
J M
JM
Smith
S J
SJ
Benkovic
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019877:Hydroxymethyl and Formyl Transferases; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010449:Peptide Mapping; D051026:Phosphoribosylglycinamide Formyltransferase; D011994:Recombinant Proteins / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C065669:N(10)-bromoacetyl-5,8-dideazafolic acid; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D000217:Acyltransferases