Ring opening is not rate-limiting in the GTP cyclohydrolase I reaction

GTP cyclohydrolase I catalyzes a mechanistically complex ring expansion aff ording dihydroneopterin triphosphate and formate from GTP, Single turnover quenched flow experiments were performed with the recombinant enzyme from E scherichia coli, The consumption of GTP and the formation of 5-formylamino- 6-ribosylamino-2-amino-4(3H)-pyrimidinone triphosphate, formate, and dihydr oneopterin triphosphate were determined by high pressure liquid chromatogra phy analysis. A kinetic model comprising three consecutive unimolecular ste ps was used for interpretations where the first intermediate, 5-formylamino -6-ribosylamino-2-amino-4 (3H)-pyrimidinone 5'-triphosphate, was formed in a reversible reaction. The rate constant k(1) for the reversible opening of the imidazole ring of GTP was 0.9 s(-1), the rate constant k(3) for the re lease of formate from 5-formylamino-6-ribosylamino-2-amino-4(3N)-pyrimidino ne triphosphate was 2.0 s(-1), and the rate constant k(1) for the formation of dihydroneopterin triphosphate was 0.03 s(-1). Thus, the hydrolytic open ing of the imidazole ring of GTP is rapid by comparison with the overall re action.