IngentaConnect UDP-galactose 4-epimerase from Kluyveromyces fragiliscatalytic si...

Abstract:

UDP-galactose 4-epimerase from Kluyveromyces fragilis is a homodimer containing one catalytic site and one NAD⁺ as cofactor per subunit. One 5′-UMP, a competitive inhibitor, binds per dimer of epimerase as isolated and causes inactivation. Addition of 0.2 mminhibitor to the enzyme in vitro leads to three sequential steps: first, the inhibitor binds to the unoccupied site; second, the inhibitor bound ex vivo is displaced allosterically; and finally, both sites are occupied by the inhibitor. These reactions have been monitored by kinetic lag in substrate conversion, coenzyme fluorescence, protection against trypsin digestion, and reductive inhibition. The transition profiles indicate the existence of a stable intermediate with one inhibitor-binding site remaining unoccupied. Reductive inhibition of this intermediate reduced the activity to 58% ± 2%, with modification of one catalytic site. A change of conformation of the epimerase upon binding with substrate or inhibitor was evident from fluorescence emission spectra. The epimerase demonstrated a biphasic Michaelis-Menten dependency. The epimerase devoid of 5′-UMP showed a Michaelis-Menten dependency that can be explained by assuming simultaneous operation of two catalytic sites. A monomeric form of the epimerase was devoid of such regulation. The inhibitory profile of 5′-UMP also suggested negative cooperativity. Incubation of the epimerase with combinations of substrate analogs rendered one of the sites inactive, supporting the presence of two functional and regulated catalytic sites. Dissimilar kinetic patterns of the reconstituted enzyme after treatment with p-chloromercuribenzoate indicated stability of the dimeric enzyme against fast association-dissociation, which could otherwise generate multiple forms of the enzyme with functional heterogeneity.

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UDP-galactose 4-epimerase from Kluyveromyces fragilis - catalytic sites of the homodimeric enzyme are functional and regulated