13C and deuterium isotope effects suggest an aldol cleavage mechanism for L-ribulose-5-phosphate 4-epimerase.
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.
Lee
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Institute for Enzyme Research, Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53705, USA.
Vu
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Cleland
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D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000737:chemistry / Q000378:metabolism; D000447:Aldehydes / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002247:Carbon Isotopes; D002384:Catalysis; D002942:Circular Dichroism; D003903:Deuterium / Q000378:metabolism*; D004098:Dihydroxyacetone / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010428:Pentosephosphates / Q000737:chemistry / Q000378:metabolism; D012274:Ribulosephosphates / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013237:Stereoisomerism; D013816:Thermodynamics
D000447:Aldehydes; D002247:Carbon Isotopes; D010428:Pentosephosphates; D012274:Ribulosephosphates; C031524:ribulose 5-phosphate; C031625:xylulose-5-phosphate; C001308:glycolaldehyde phosphate; C116609:3-hydroxybutanal; D003903:Deuterium; D000446:Aldehyde-Lyases; C036027:L-fuculosephosphate aldolase; C022526:rhamnulose-1-phosphate adolase; D002238:Carbohydrate Epimerases; C022554:L-ribulosephosphate 4-epimerase; D000079:Acetaldehyde; D004098:Dihydroxyacetone