The Mechanism of Action of the Enzyme Uridine Diphosphoglucose 4-Epimerase

Abstract

Abstract The properties of the enzyme UDP-glucose 4-epimerase from Escherichia coli have been investigated. Its substrate specificity is comparatively low, it acts on the thymidine, cytidine, and adenine, as well as the uridine diphospho derivatives of glucose. Its specificity toward modifications of carbon 6 of the hexose moiety of the substrate is also low. The formation of NADH on the enzyme in the presence of substrate (Wilson, D. B., and Hogness, D. S., J. Biol. Chem., 239, 2469 (1964)) is less specific for the substrate than is the over-all epimerization. UDP-mannose causes reduction of the enzyme-bound NAD+ but is not epimerized. It has been shown that the epimerization is an oxidation-reduction process involving NAD+ as the oxidant and UDP-4-ketoglucose as the enzyme-bound oxidized intermediate. Transfer of the 4-hydrogen from the hexose moiety of UDP-6-deoxyglucose to the B-position of NADH and its transfer back to several nucleotide-4-keto sugars has been demonstrated. The nucleotide-4-keto sugars were synthesized using enzymatic or chemical reactions or both. The enzyme-bound NAD+ is subject to reduction by NaBH4 and the hydrogen is specifically transferred to the B-side of the nicotinamide ring. When conducted in the presence of substrate, this chemical reduction of NAD+ is accompanied by the binding of 1 mole of the substrate and complete loss of enzyme activity. Another method of reducing the enzyme-bound NAD+ is to incubate the enzyme with substrate for long periods of time. In addition to the steady state level, there is a slow increase in enzyme-NADH which is accompanied by loss of enzyme activity, strong binding of 1 mole of substrate and incorporation of the 4-hydrogen of the substrate into the B-position of NADH. Both forms of the reduced enzyme can be oxidized and reactivated by nucleotide-4-keto sugars with removal of the B-hydrogen from NADH. The possibility that tryptophan functions as an intermediate hydrogen carrier in this reaction, as it has been proposed to do in several other NAD+-linked dehydrogenase reactions (Schellenberg, K. A., J. Biol. Chem., 242, 1815 (1967)), has been investigated. It would appear that there are no intermediate hydrogen carriers involved.