UDP-galactose 4-epimerase from Kluyveromyces fragilis – catalytic sites of the homodimeric enzyme are functional and regulated
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UDP-galactose 4-epimerase from Kluyveromyces
fragilis – catalytic sites of the homodimeric enzyme
are functional and regulated
Structural Biology & Bioinformatics
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 mm inhibitor 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
Brahma, Amrita and Banerjee, Nupur and Bhattacharyya, Debasish (2009) UDP-galactose 4-epimerase from Kluyveromyces fragilis – catalytic sites of the homodimeric enzyme are functional and regulated. FEBS Journal, 276 (22). pp. 6725-6740.