13C and deuterium isotope effects suggest an aldol cleavage mechanism for L-ribulose-5-phosphate 4-epimerase.

On the basis of 13C and deuterium isotope effects, l-ribulose-5-phosphate 4-epimerase catalyzes the epimerization of l-ribulose 5-phosphate to d-xylulose 5-phosphate by an aldol cleavage to the enediolate of dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group and condensation to the epimer at C-4. With the wild-type enzyme, 13C isotope effects were 1.85% at C-3 and 1.5% at C-4 at pH 7, with the values increasing to 2.53 and 2.05% at pH 5.5, respectively. H97N and Y229F mutants at pH 7 gave values of 3.25 and 2.53% at C-3 and 2.69 and 1.99% at C-4, respectively. Secondary deuterium isotope effects at C-3 were 2.5% at pH 7 and 3.1% at pH 5.5 with the wild-type enzyme, and 4.1% at pH 7 with H97N. At C-4, the corresponding values were 9.6, 14, and 19%. These data suggest that H97N shows no commitments, while the wild-type enzyme has an external commitment of ∼1.4 at pH 7 and an internal commitment independent of pH of ∼0.6. The Y229 mutant shows only the internal commitme...