Subcloning, Characterization, and Affinity Labeling of Escherichia coli Glycinamide Ribonucleotide Transformylase

James Inglese, Dana L. Johnson, Stephen J. Benkovic, Alan Shiau, John M. Smith

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

Abstract

Glycinamide ribonucleotide transformylase (GAR TFase; EC 2.1.2.2) has been purified 70-fold to apparent homogeneity from Escherichia coli harboring an expression vector encoding the purN gene product, GAR TFase. The protein is a monomer of Mr 23 241 and catalyzes a single reaction. Steady-state kinetic parameters for the enzyme have been obtained. The structural requirements for cofactor utilization have been investigated and found to parallel those of the multifunctional avian enzyme. The enzyme was inactivated with the affinity label N10-(bromoacetyl)-5,8-dideazafolate in a stoichiometric and activesite-specific manner. The ionization state of the cofactor analogue in the enzyme-cofactor complex appears to require the dissociation of the proton at N3 of the pyrimidine within the complex.

Original languageEnglish (US)
Pages (from-to)1436-1443
Number of pages8
JournalBiochemistry
Volume29
Issue number6
DOIs
StatePublished - Feb 1 1990

All Science Journal Classification (ASJC) codes

  • Biochemistry

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