Yeast orotidine-5'-phosphate decarboxylase: steady-state and pre-steady-state analysis of the kinetic mechanism of substrate decarboxylation.

The catalytically active form of monofunctional yeast orotidine-5'-phosphate decarboxylase was a dimer (E(2)). The dimer equilibrium dissociation constant was 0.25 microM in 0.01 M MOPS Na(+) at pH 7.2. The bimolecular rate constant for dimer formation was 1.56 microM(-1) s(-1). The dimeric form of the enzyme was stabilized by NaCl such that the enzyme was E(2) in 100 mM NaCl at all concentrations of enzyme tested. The kinetics of binding of OMP to E(2) was governed by two ionizations (pK(1) = 6.1 and pK(2) = 7.7). From studies with substrate analogues, the higher pK was assigned to a group on the enzyme that interacted with the pyrimidinyl moiety. The value of the lower pK was dependent on the substrate analogue, which suggested that it was not exclusively the result of ionization of the phosphoryl moiety. During the decarboxylation of OMP, the fluorescence of E(2) was quenched over 20%. The enzymatic species with reduced fluorescence was a catalytically competent intermediate that had kinetic properties consistent with it being the initial enzyme-substrate complex. The stoichiometry for binding of OMP to E(2) was one OMP per enzyme monomer. The value of the first-order rate constant for conversion of the enzyme-substrate complex to free enzyme (36 s(-1)) calculated from a single turnover experiment ([E] >> [S]) was slightly greater than the value of k(cat), 20 s(-1) (corrected for stoichiometry), calculated from steady-state data. In the single turnover experiments, the enzyme was E(2)*S, whereas in the steady-state turnover the experiment enzyme was E(2)*S(2). The similarity of these values suggested that the subunits were catalytically independent such that E(2)*S(2) could be treated as E*S and that conversion of the enzyme-substrate complex to E was k(cat). Kinetic data for the approach to the steady-state with OMP and E(2) yield a bimolecular association rate complex of 62 microM(-1) s(-1)and a dissociation rate constant for E*S of 60 s(-1). The commitment to catalysis was 0.25. By monitoring the effect of carbonic anhydrase on [H(+)] changes during a single turnover experiment, the initial product of the decarboxylation reaction was shown to be CO(2) not HCO(3-). UMP was released from the enzyme concomitantly with CO(2) during the conversion of E*S to E. Furthermore, the enzyme removed an enzyme equivalent of H(+) from solvent during this step of the reaction. The bimolecular rate constants for association of 6-AzaUMP and 8-AzaXMP, substrate analogues with markedly different nucleobases, had association rate constants of 112 and 130 microM(-1) s(-1), respectively. These results suggested that the nucleobase did not contribute significantly to the success of formation of the initial enzyme-substrate complex.