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

On the basis of C-13 and deuterium isotope effects, L-ribulose-5-phosphate 4-epimerase catalyzes the epimerization of L-ribulose 5-phosphate to D-xylu lose 5-phasphate by an aldol cleavage to the enediolate of dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group a nd condensation to the epimer at C-4. With the wild-type enzyme. C-13 isoto pe effects were 1.85% at C-3 and 1.5% at C-4 at pH 7, with the values incre asing 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, respec tively. 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 H9 7N shows no commitments, while the wild-type enzyme has an external commitm ent of similar to 1.4 at pH 7 and an internal commitment independent of pH of similar to 0.6. The Y229 mutant shows only the internal commitment of 0. 6. The sequence of the epimerase is similar to those of L-fuculose-1-phosph ate and L-rhamnulose-1-phosphate aldolases for residues in the active site of L-fuculose-1-phosphate aldolase, suggesting that Asp76, His95, His97, an d His171 of the epimerase may be metal ion ligands, and Ser44, Gly45, Ser74 , and Ser75 may form a phosphate binding pocket. The pH profile of V/K for L-ribulose 5-phosphate is bell-shaped with pK values of 5.94 and 8.24. The CD spectra of L-ribulose 5-phasphate and D-xylulose 5-phosphate differ suff iciently that the epimerization reaction can be followed at 300 nm.