Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is the locus of
Lesch-Nyhan syndrome, the activator of the
prodrugs 6-mercaptopurine and
allopurinol, and a target for
antiparasitic chemotherapy. The three-dimensional structure of the recombinant human
enzyme in complex with GMP has recently been solved [Eads, J., Scapin, G., Xu, Y., Grubmeyer, C., & Sacchettini, J. C. (1994) Cell 78, 325-334]. Here,
ligand binding, pre-steady state kinetics,
isotope trapping, and
isotope exchange experiments are presented which detail the sequential kinetic mechanism of the
enzyme. In the forward reaction, in which a base (
hypoxanthine or
guanine) reacts with PRPP to form
nucleoside monophosphate and PPi, binding of PRPP precedes that of the base, and in the reverse direction,
IMP binds first. Compared to k(cat), phosphoribosyl group transfer is rapid in both the forward (131 vs 6.0 s(-1)) and reverse (9 vs 0.17 s(-1)) directions. In the forward direction, product
pyrophosphate dissociates rapidly (> 12 s(-1)) followed by release of
IMP (6.0 s(-1)). In the reverse direction, Hx dissociates rapidly (9.5 s(-1)) and PRPP dissociates slowly (0.24 s(-1)). The more rapid rate of utilization of
guanine than
hypoxanthine in the forward reaction is the result of the faster release of product GMP rather than the result of differences in the rate of the chemical step. The kinetic mechanism, with rapid chemistry and slow product dissociation, accounts for the previously observed ability of the alternative product
guanine to stimulate, rather than inhibit, the pyrophosphorolysis of
IMP. The overall equilibrium for the
hypoxanthine phosphoribosyl transfer reaction lies far toward
nucleotide product (Keq approximately 1.6 x 10(5)), at the high end for PRPP-linked
nucleotide formation. The three-dimensional structure of the HGPRTase x
IMP complex has been solved to 2.4 A resolution and is isomorphous with the GMP complex. The results of the
ligand binding and kinetic studies are discussed in light of the structural data.