Figure 8.

Free energy reaction profiles that illustrate the different effects of the R235A mutation on the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP to form UMP (profile on left) and the deuterium exchange reaction of h-FUMP to form d-FUMP (profile on right). The binding energy of R235A and other residues in the phosphodianion gripper loop is utilized to drive the conversion of open enzyme E_O to closed enzyme E_C – this binding energy is partly expressed at the Michaelis complex when loop closure at the Michaelis complex is thermodynamically favorable (K_c > 1, eq 4 – 6). The substrate binding energy (ΔG_S)_wt of OMP is greater than for FUMP, and we propose that this reflects the utilization of the binding interactions with the -CO₂⁻ group of OMP to drive the conversion of E_O to E_C (ΔG’_c). The overall effect of the R235A mutation on the barrier for the deuterium exchange (ΔΔG^‡_UMP = 7.2 kcal/mol) is larger than for the decarboxylation reaction (ΔΔG^‡_OMP = 5.6 kcal/mol). The R235A mutation results mainly in a decrease in ΔG_S for the decarboxylation reaction (ΔΔG_S = 4 kcal/mole). We propose that the Michaelis complex between OMP and the R235A mutant enzyme exists mainly in the nonproductive open form ([Eo•OMP]_R235A), because there is insufficient binding energy at this Michaelis complex enzyme to drive the enzyme conformational change (K_c < 1, eq 4–6). The effect of the R235A mutation on k_cat (ΔΔG^‡_kcat = 1.6 kcal/mol) is shown as the 1.6 kcal/mol barrier for conversion of [Eo•OMP]_R235A to the productive complex ([E_C•OMP]_R235A). The smaller binding energy from FUMP compared with OMP available to drive the enzyme conformational change ((K_c)_FUMP < (K_c)_OMP, eq 4 – 6) results in the smaller the effect of the R235A mutation on ΔG_S (ΔΔG_S = 2.2 kcal/mol) and a large 5 kcal/mol barrier to conversion of nonproductive [Eo•FUMP]_R235A to productive ([E_C•FUMP]_R235A that is added to the barrier for conversion of [Eo•FUMP]_R235A to product (ΔΔG^‡_kcat = 5 kcal/mol).