Enzyme Architecture: Breaking Down the Catalytic Cage that Activates Orotidine 5'-Monophosphate Decarboxylase for Catalysis.

We report the results of a study of the catalytic role of a network of four interacting amino acid side chains at yeast orotidine 5'-monophosphate decarboxylase (ScOMPDC), by the stepwise replacement of all four side chains. The H-bond, which links the -CH2 OH side chain of S154 from the pyrimidine umbrella loop of ScOMPDC to the amide side chain of Q215 in the phosphodianion gripper loop, creates a protein cage for the substrate OMP. The role of this interaction in optimizing transition state stabilization from the dianion gripper side chains Q215, Y217 and R235 was probed by determining the kinetic parameter kcat /Km for 16 enzyme variants, which include all combinations of single, double, triple and quadruple mutations of S154A Q215A, Y217F and R235A. The effects of consecutive Q215A, Y217F and R235A mutations on ΔG‡ for wildtype enzyme-catalyzed decarboxylation sum to 11.6 kcal/mol, but to only 7.6 kcal/mol, when starting from S154A mutant. This shows that the S154A mutation results in a (11.6 - 7.6) = 4.0 kcal/mol decrease in transition state stabilization from interactions with Q215, Y217 and R235. Mutant cycles show that ca 2 kcal/mol of this stabilization at wildtype ScOMPDC is from the direct interaction of the Q215 side chain and that ca. 2 kcal/mol is from a tightening in the interactions with Y217 and R235. The sum of the effects of single S154, Q215, Y217 and R235 substitutions at the quadruple mutant of ScOMPDC on ΔG‡ , 5.4 kcal/mol, is much smaller than 16.0 kcal/mol, the sum of the effects of the related four substitutions at wildtype ScOMPDC to give the respective single mutants. The former small effects are consistent with a large entropic cost to holding the flexible loops of ScOMPDC in the active closed conformation.