Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies.
Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E. , Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281, 485-499], permitting a more exact formulation of the probable catalytic mechanism.
Shim
J H
JH
Department of Chemistry, The Pennsylvania State University, University Park 16802, USA.
Benkovic
S J
SJ
D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017666:Deuterium Oxide; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D051026:Phosphoribosylglycinamide Formyltransferase; D010957:Plasmids / Q000138:chemical synthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012997:Solvents
D011994:Recombinant Proteins; D012997:Solvents; C092738:10-formyl-5,8-dideazafolate; D005492:Folic Acid; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D017666:Deuterium Oxide