| Abstract
| - On the basis of 13C and deuterium isotope effects, l-ribulose-5-phosphate 4-epimerase catalyzesthe epimerization of l-ribulose 5-phosphate to d-xylulose 5-phosphate by an aldol cleavage to the enediolateof dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group andcondensation to the epimer at C-4. With the wild-type enzyme, 13C isotope effects were 1.85% at C-3 and1.5% at C-4 at pH 7, with the values increasing to 2.53 and 2.05% at pH 5.5, respectively. H97N andY229F 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-typeenzyme, and 4.1% at pH 7 with H97N. At C-4, the corresponding values were 9.6, 14, and 19%. Thesedata suggest that H97N shows no commitments, while the wild-type enzyme has an external commitmentof ∼1.4 at pH 7 and an internal commitment independent of pH of ∼0.6. The Y229 mutant shows onlythe internal commitment of 0.6. The sequence of the epimerase is similar to those of l-fuculose-1-phosphateand l-rhamnulose-1-phosphate aldolases for residues in the active site of l-fuculose-1-phosphate aldolase,suggesting that Asp76, His95, His97, and 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-ribulose5-phosphate is bell-shaped with pK values of 5.94 and 8.24. The CD spectra of l-ribulose 5-phosphateand d-xylulose 5-phosphate differ sufficiently that the epimerization reaction can be followed at 300 nm.
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