Mechanistic aspects of carbohydrate epimerization

• Main Author: Samuel, Jomy
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/14895

Epimerases are enzymes that invert the stereochemistry at a stereogenic center in a molecule with two or more chiral centers. Most known sugar epimerases catalyze this reaction by simple deprotonation/reprotonation or by oxidation/reduction mechanism. Only two carbohydrate epimerases are known that catalyze the inversion of stereochemistry by mechanisms different from those mentioned above. These are: L-ribulose-5-phosphate 4- epimerase and UDP-7V-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase). L-Ribulose-5-phosphate 4-epimerase (L-Ru5P 4-epimerase) is a bacterial enzyme that interconverts L-ribulose 5-phosphate (L-Ru5P) and D-xylulose 5-phosphate (D-Xu5P) by a retroaldol/aldol mechanism. The epimerase shares 26% sequence homology and a high degree of structural homology with the metal-dependent class-II aldolase, L-fuculose-1 -phosphate aldolase, which catalyzes the reversible C-C bond cleavage of L-fuculose 1-phosphate (LFulP) to give L-lactaldehyde and dihydroxyacetone phosphate. We have shown that the epimerase and the aldolase share a conserved phosphate binding pocket. Since the substrates of the epimerase and the aldolase are phosphorylated at opposite ends, the epimerase binds the substrate in a reverse orientation as compared to the aldolase. Due to this "flipped" orientation of the bound substrates, the two enzymes utilize different acid/base residues for catalyzing the reaction. Asp 120' has been identified as the catalytic residue responsible for deprotonating DXu5P. Thus, while there is a single catalytic residue, Glu73, in the aldolase, there are two different catalytic residues, Aspl20' and Tyr229' (Cleland et al.)64 in the epimerase. UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase or UDPE) is a homodimeric, bacterial enzyme that catalyzes the interconversion of UDP-N- acetylglucosamine (UDP-GlcNAc) and UDP-N-acety1mannosamine (UDP-ManNAc) by a mechanism involving C-0 bond cleavage/formation. UDPE is allosterically regulated by its own substrate UDP-GlcNAc. Using site-directed mutagenesis we have identified two residues, His213 and Lysl5, which play an important role in substrate binding. Three carboxylate mutants, D95N, E117Q and E131Q, showed a 10,000-fold decrease in catalytic efficiency and an impaired allosteric control. The ability of the E117Q mutant to catalyze the release of intermediates from UDP-GlcNAc at rates comparable to that of the wild-type enzyme suggests that Glul l7 could be the catalytic residue responsible for deprotonating UDP-ManNAc. The wild-type epimerase shows a 50% increase in fluorescence with 1 mM of a 10:1 mixture of UDP-GlcNAc and UDP-ManNAc. On the basis of kinetic and binding studies with the wild-type epimerase and the three carboxylate mutants we have shown that the enzyme is capable of binding UDP-ManNAc in the absence of UDP-GlcNAc. We propose that UDPE is a V-system in which binding of UDP-GlcNAc to one subunit increases the catalytic efficiency of the other subunit.