Active-Site Mapping and Site-Specific Mutagenesis of Glycinamide Ribonucleotide Transformylase from Escherichia coli

James Inglese, John M. Smith, Stephen J. Benkovic

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46 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)6678-6687
Number of pages10
JournalBiochemistry
Volume29
Issue number28
DOIs
StatePublished - Jul 1 1990

All Science Journal Classification (ASJC) codes

  • Biochemistry

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