Reactions of phosphorothioate compounds catalyzed by adenylosuccinate synthetase. Steady state and pre-steady state kinetic studies.

Kinetic studies of the reactions catalyzed by adenylosuccinate synthetase with phosphorothioate derivatives of substrates for the forward and reverse reactions provide evidence for an intermediate on the reaction pathway. Guanosine-5’-0-(3-thiotriphosphate) (GTPyS) replaces GTP with a 12.5-fold reduction in the maximal velocity (at 22”C), although the K, for GTPyS is 3.5-fold lower than the K,,, for GTP. Comparison of the steady state kinetic data for the GTPyS and GTP reactions indicates that for the reaction with GTP the kinetic mechanism is not rapid equilibrium for all substrates. When thiophosphate replaces phosphate in the reverse reaction, the principal products are GTP, 6mercaptopurine ribotide, and aspartate. The rate of the thiophosphate reaction is 14% of the rate obtained for the reaction with phosphate. When the reaction with GTPyS was monitored at 3°C the approach of the reaction to a steady state rate could be observed. Adenylosuccinate formation exhibited a lag phase before attainment of the steady state rate whereas IMP disappearance showed a burst phase. The initial rate of disappearance of IMP is identical for reactions with CaGTP, MgGTPyS, and CaGTPyS even though the steady state rates vary by lOO-fold. Substrate disappearance and product formation curves are biphasic for MgGTPyS, CaGTP, and CaGTPyS indicating the presence of an intermediate between substrate and product in these reactions. The reaction with MgGTP shows equivalent monophasic rate profiles for product formation and substrate disappearance. The steady state turnover rate with MgGTP is equal to the rate constant for the initial phase of substrate disappearance with MgGTPyS, CaGTP, and CaGTPyS. The monophasic kinetics observed with MgGTP are consistent with the presence of an intermediate whose formation is rate-limiting, and pre-steady state kinetic measurements of the reverse reaction are also consistent with this interpretation. Ultraviolet difference spectra recorded during the transient phase of the reaction indicate that the intermediate does not have a strong absorption in the ultraviolet. The kinetic and spectral data suggest that the intermediate may be an addition product between IMP and aspartate.