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 k inetic parameters of the mutants has been used to probe the role of the act ive site residues and loops in catalysis by glycinamide ribonucleotide tran sformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conser ved 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 geom etry 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 iso tope effect of 2. The ionization of the amino group of glycinamide ribonucl eotide 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 o f the substrate. The kinetic behavior parallels pH effects revealed by a se ries of X-ray crystallographic structures of the apoenzyme and inhibitor-bo und 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. Mel. Biol. 281, 485-499], permitting a more exact formulation of the probable ca talytic mechanism.