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L-ribulose-5-phosphate 4-epimerase: epimerisation through carbon-carbon bond cleavage

University of British Columbia

L-Ribulose-5-phosphate 4-epimerase, a bacterial enzyme which catalyses the final step in L-arabinose metabolism, interconverts L-ribulose-5-phosphate and D-xylulose-5-phosphate independently of NAD+. The labile stereocentre does not bear an acidic proton; therefore, a simple deprotonation-reprotonation mechanism cannot be followed. The epimerase was cloned from Escherichia coli into a highly efficient overexpression vector. The recombinant enzyme was found to contain a mixture of divalent zinc, manganese and copper ions. A preparation of a homogeneous sample of the Zn²+ form of the recombinant enzyme displayed kinetic constants similar to those of the naturally abundant epimerase from E. coli. Amino acid sequence similarity between the epimerase and the Class II L-fuculose-1- phosphate aldolase suggests that these two enzymes may be evolutionarily related and that the epimerisation, which is metal-dependent, may occur through carbon-carbon bond cleavage and reformation. Three conserved residues (H95, H97 and D76) which are thought to be the metal ion ligands in the epimerase have been independently altered by site directed mutagenesis to asparagine. The resulting mutant epimerases exhibit low kcat values. The H95N and H97N epimerases have a reduced affinity for Zn²+ and lose metal readily, while the D76N epimerase which has an affinity for Zn²+ comparable to that of the wild type epimerase loses metal upon extended dialysis. These observations serve to establish a structural link between the active sites of the epimerase and the aldolase. The H97N epimerase was found capable of catalysing the aldol addition between dihydroxyacetone and glycolaldehyde phosphate (the unbound forms of the proposed reaction intermediates) to form an equilibrium mixture of L-ribulose-5-phosphate and Dxylulose- 5-phosphate. In addition, the epimerase was able to release dihydroxyacetone from an equiHbrating pool of L-ribulose-5-phosphate and D-xylulose-5-phosphate. These observations of aldolase activity establish that the active site of the epimerase is capable of catalysing carbon-carbon bond cleavage, and support the notion that the epimerase and the aldolase are evolutionarily related. Glycolaldehyde phosphate was shown to be a competitive inhibitor of the H97N enzyme with a KI of 0.37 mM. The wild type enzyme was not significantly inhibited at 5 mM. The H97N mutation appears to have created a "leaky" epimerase which can bind to the normal reaction intermediates and generate them from the unbound aldol cleavage products. [Scientific formulae used in this abstract could not be reproduced.]

Thesis/Dissertation

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2009-06-03

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http://hdl.handle.net/2429/8686

Chemistry

University of British Columbia

UBCV

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