PMID:2204419
| Citation |
Inglese, J, Smith, JM and Benkovic, SJ (1990) Active-site mapping and site-specific mutagenesis of glycinamide ribonucleotide transformylase from Escherichia coli. Biochemistry 29:6678-87 |
|---|---|
| Abstract |
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. |
| Links | |
| Keywords |
Acyltransferases/antagonists & inhibitors; Acyltransferases/genetics; Acyltransferases/metabolism; Amino Acid Sequence; Bacterial Proteins/genetics; Base Sequence; Binding Sites; Escherichia coli/enzymology; Escherichia coli/genetics; Folic Acid/analogs & derivatives; Folic Acid/metabolism; Folic Acid/pharmacology; Hydroxymethyl and Formyl Transferases; Molecular Sequence Data; Oligodeoxyribonucleotides/chemical synthesis; Peptide Mapping; Phosphoribosylglycinamide Formyltransferase; Recombinant Proteins/genetics; Sequence Homology, Nucleic Acid; Tetrahydrofolate Dehydrogenase/genetics |
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