UBC Theses and Dissertations
The Mechanism of UDP-N-acetylglucosamine 2-epimerase Morgan, Paul M.
The bacterial enzyme UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2- epimerase) catalyzes the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc), by inverting the stereochemistry at C-2" of the nucleotide sugars. The mechanism of this enzyme is interesting, because the proton at the C-2" position is not acidic, and therefore the reaction must proceed via a pathway that is more complicated than a simple deprotonation followed by reprotonation in the inverted stereochemical sense. A coupled enzyme assay for UDP-GlcNAc 2-epimerase activity has been developed using UDP-ManNAc dehydrogenase, an enzyme that catalyzes the NAD⁺-dependent two-fold oxidation of UDP-ManNAc to form UDP-N-acetylmannosaminuronic acid. In order to obtain the substantially large amounts of the dehydrogenase required for the coupled assay, it was necessary to clone and overexpress the enzyme from Escherichia coli. The recombinant dehydrogenase was purified to homogeneity, and determined to have a kcat of 1.2 ± 0.2 s⁻¹ and an apparent Km of 1.2 ± 0.4 mM under the conditions relevant to the coupled assay (at pH 8.8). The coupled enzyme assay permitted the kinetic characterization of recombinant E. coli UDP-GlcNAc 2-epimerase. The epimerase was purified to homogeneity and determined to have a kcat of 4.8 ± 0.2 s"1, and an apparent K m of 0.73 ± 0.09 mM. The epimerase also displays positive cooperativity, with a Hill coefficient of 2.3 ± 0.2. These values agree with those reported previously (Kawamura et al., 1979). The enzymatic epimerization in D₂O proceeds with the incorporation of deuterium into the C-2" position, supporting a mechanism that ultimately involves proton transfer at this position. The epimerization with 2"- ²H-UDP-GlcNAc as the substrate is slowed by a primary kinetic isotope effect (kH/kD = 1.8 ± 0.1) indicating that the C-H bond at C-2" is cleaved during a rate determining step of the reaction. The enzyme does not require the addition of any NAD⁺ cofactor for activity, and experiments failed to detect any cofactor tightly bound within the enzyme, suggesting that NAD⁺ is not involved in the epimerization mechanism. The enzyme is also observed to slowly release UDP and 2-acetamidoglucal when a large quantity of epimerase is incubated with substrate over extended periods. These observations are consistent with an unprecedented enzymatic epimerization mechanism that proceeds via cleavage of the anomeric C-O bond. The simplest reasonable description of the enzyme mechanism involves the anti elimination of UDP from UDP-GlcNAc, to form 2- acetamidoglucal and UDP as reaction intermediates, followed by the syn addition of UDP to the glycal double bond. Similarly, the reverse reaction requires the syn elimination of UDP from UDP-ManNAc coupled with the anti addition of UDP to the 2-acetamidoglucal intermediate. An experiment to identify the general base or bases involved in the deprotonation of the C-2" proton required the synthesis of uridine 5'-diphosphate 2,3-epoxypropanol (UDPglycidol), an affinity label specific for the UDP-GlcNAc 2-epimerase active site. However, under the experimental conditions, the covalent-labeling compound failed to inactivate the epimerase in an irreversible fashion. UDP-Glycidol was found, however, to irreversibly inactivate UDP-ManNAc dehydrogenase, and a specific cysteine residue is implicated as the site of covalent modification of the enzyme.
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