¹³C and Deuterium Isotope Effects Suggest an Aldol Cleavage Mechanism for l-Ribulose-5-phosphate 4-Epimerase

Abstract

On the basis of (13)C 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, (13)C 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 approximately 1.4 at pH 7 and an internal commitment independent of pH of approximately 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-phosphate and 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-ribulose 5-phosphate is bell-shaped with pK values of 5.94 and 8.24. The CD spectra of L-ribulose 5-phosphate and D-xylulose 5-phosphate differ sufficiently that the epimerization reaction can be followed at 300 nm.

Enzymatic Characterization of 5-Methylthioribulose-1-phosphate Dehydratase of the Methionine Salvage Pathway inBacillus subtilis

“…8). 23) In addition, MtnB is a homotetramer, as are the FucA, RibE, and RhuA enzymes. 14,18,24) These similarities suggest that MtnB is a new member of the FucA/ RibE/RhuA family and that these enzymes are related to each other evolutionally.…”

The Use of Isotope Effects to Determine Enzyme Mechanisms

“…Lac Lee showed the power of isotope effects to deduce mechanism in his studies of L-ribulose-5-P 4-epimerase (42). He devised a way to degrade the xylulose-5-P product carbon by carbon and showed that there were over 2% 13 C isotope effects at C-3 and C-4.…”

The Chemical Mechanism of D‐1‐Deoxyxylulose‐5‐phosphate Reductoisomerase from Escherichia coli

“…[19] This enzyme interconverts l-ribulose-5-phosphate and d-xylulose-5-phosphate, but requires a divalent metal ion. DXR appears to catalyze a very similar reaction, albeit slowly, in the absence of a metal.…”