Kinetic mechanism of OMP synthase: A slow physical step following group transfer limits catalytic rate

Orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10) forms the UMP precursor orotidine 5'-monophophate (OMP) from orotate and alpha-D-5-phosp horibosyl-1-pyrophosphate (PRPP). Here, equilibrium binding, isotope partit ioning, and chemical quench studies were used to determine rate and equilib rium constants for the kinetic mechanism. PRPP bound to two sites per dimer with a K-D of 33 mu M. Binding of OMP and orotate also occurred to a singl e class of two sites per dimer, with K-D values of 3 and 280 mu M, respecti vely. Pyrophosphate binding to two sites was weak with a K-D of 960 mu M, a nd in the presence of bound orotate, its affinity for the first site was en hanced 4-fold (K-D = 230 mu M). Preformed E.OMP, E.PRPP, E.PPi, and E.orota te complexes were trapped as products in isotope partitioning experiments, indicating that each was catalytically competent and confirming a random me chanism. Rapid quench experiments revealed burst kinetics for product forma tion in both the forward phosphoribosyltransferase and the reverse pyrophos phorolysis reactions. The steady-state rate in the forward reaction was pre ceded by a burst (n(fwd) = 1.5/dimer) of at least 300 s(-1). In the pyropho sphorolysis reaction, a burst (n(rev) = 0.7/dimer; k greater than or equal to 300 s(-1)) was also noted. These results allowed us to develop a complet e kinetic mechanism for OPRTase, in which a rapid phosphoribosyl transfer r eaction at equilibrium is followed by a slow step involving release of prod uct. When the microviscosity, eta(rel), of the reaction medium was increase d with sucrose, the forward k(cat) decreased in proportion to eta(rel) with a slope of 0.8. In the reverse reaction a more limited dependence of k(cat ) (slope = 0.3) was observed. On the basis of the known structures of OPRTa se, we propose that a highly conserved, catalytically important, solvent-ex posed loop descends during catalysis to shield the active site. In the acco mpanying paper, the slow product release step is shown to relate to movemen t of the solvent-exposed loop.