Orotidine 5′-Monophosphate Decarboxylase: The Operation of Active Site Chains Within and Across Protein Subunits

Abstract

The D37 and T100′ side chains of orotidine 5′-monophosphate decarboxylase (OMPDC) interact with the C-3′ and C-2′ ribosyl hydroxyl groups, respectively, of the bound substrate. We compare the intra-subunit interactions of D37 with the inter-subunit interactions of T100′ by determining the effects of the D37G, D37A, T100′G, and T100′A substitutions on the following: (a) k cat and k cat / K m values for the OMPDC-catalyzed decarboxylations of OMP and 5-fluoroorotidine 5′-monophosphate (FOMP) and (b) the stability of dimeric OMPDC relative to the monomer. The D37G and T100′A substitutions resulted in 2 kcal mol –1 increases in Δ G † for k cat / K m for the decarboxylation of OMP, while the D37A and T100′G substitutions resulted in larger 4 and 5 kcal mol –1 increases, respectively, in Δ G † . The D37G and T100′A substitutions both resulted in smaller 2 kcal mol –1 decreases in Δ G † for the decarboxylation of FOMP compared to that of OMP. These results show that the D37G and T100′A substitutions affect the barrier to the chemical decarboxylation step while the D37A and T100′G substitutions also affect the barrier to a slow, ligand-driven enzyme conformational change. Substrate binding induces the movement of an α-helix (G′98–S′106) toward the substrate C-2′ ribosyl hydroxy bound at the main subunit. The T100′G substitution destabilizes the enzyme dimer by 3.5 kcal mol –1 compared to the monomer, which is consistent with the known destabilization of α-helices by the internal Gly side chains [Serrano, L., et al. (1992) Nature , 356 , 453–455]. We propose that the T100′G substitution weakens the α-helical contacts at the dimer interface, which results in a decrease in the dimer stability and an increase in the barrier to the ligand-driven conformational change.

Protein–Ribofuranosyl Interactions Activate Orotidine 5′-Monophosphate Decarboxylase for Catalysis

“… a At pH 7.1 (30 mM MOPS) and I = 0.105 (NaCl). b Rate constant defined in Scheme . c Published kinetic parameters d The effect of the side chain substitution on the activation barrier for the reaction catalyzed by wild-type OMPDC. e Published kinetic parameter …”

“…The side chains move >2 Å upon substrate binding and form contacts to ribosyl −OH groups that stabilize the active closed enzyme relative to the inactive open form. We report here the results of experiments designed to establish whether protein motion that positions the D37 and T100′ side chains of OMPDC to interact with the ribosyl hydroxyls of OMP plays a role in enzyme activation similar to that documented for motion of phosphodianion gripper side chains toward the bound dianion. , We previously reported the effect of D37G/A and T100′G/A substitutions on the kinetic parameters for OMPDC-catalyzed decarboxylation of OMP and FOMP and on the stability of the active enzyme dimer relative to the inactive monomer . We report here the results of experiments to determine the contribution of these side chain interactions to stabilization of the transition states for the unactivated and small sugar dianion-activated decarboxylation reactions of truncated substrates EO and FO .…”

“…All other chemicals were of reagent grade or better and were used without further purification. The procedures for the preparation, overexpression, and purification of T100′A and D37G variants of orotidine 5′-monophosphate decarboxylase from Saccharomyces cerevisiae were described in an earlier publication …”

“…12,13 We previously reported the effect of D37G/A and T100′G/A substitutions on the kinetic parameters for OMPDC-catalyzed decarboxylation of OMP and FOMP and on the stability of the active enzyme dimer relative to the inactive monomer. 14 We report here the results of experiments to determine the contribution of these side chain interactions to stabilization of the transition states for the unactivated and small sugar dianion-activated decarboxylation reactions of truncated substrates EO and FO.…”

“…The procedures for the preparation, overexpression, and purification of T100′A and D37G variants of orotidine 5′-monophosphate decarboxylase from Saccharomyces cerevisiae were described in an earlier publication. 14 Preparation of Solutions. Solution pH was determined at 25 °C using an Orion model 720A pH meter equipped with a Radiometer pHC4006-9 combination electrode that was standardized at pH 4.00, 7.00, and 10.00 at 25 °C.…”