| Summary: | The bacterial enzyme UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-
epimerase) catalyzes the interconversion between the sugar nucleotide UDP-N-acetylglucosamine
(UDP-GlcNAc) and its C-2" epimer UDP-N-acetylmarinbsamine (UDP-ManNAc).
This enzyme differs from known racemases and epimerases in that it must invert a
stereogenic center that bears a non-acidic proton. It must therefore employ a mechanism that is
more complicated than a simple inversion by deprotonation followed by reprotonation on the
opposite face of the reaction center.
Previous studies on the epimerase obtained from natural sources have revealed that the
substrate (UDP-GlcNAc) is required as an activator in the reverse reaction (UDP-ManNAc to
UDP-GlcNAc), and that the enzyme is highly specific for its normal substrates. In addition,
evidence was obtained suggesting that a proton transfer was ultimately responsible for the
epimerization. A mechanism was suggested in which the epimerization was facilitated by
increasing the acidity of the C-2" proton via transient formation of a sugar nucleotide ketointermediate
(Kawamura et al., 1978: 1979; Salo, 1976). These results have been corroborated
on a recombinant E. coli UDP-GlcNAc 2-epimerase and evidence supporting an alternate
mechanism has been obtained.
A substrate labeled with 18O at the anomeric position was prepared in order to be used in
Positional Isotope Exchange (PIX) experiments. These experiments test whether the anomeric
bond is cleaved during the epimerization. The scrambling of an 18O label located at the β-
31
phosphate moiety of UDP-epimers was detected by 31P NMR spectroscopy. This provided
strong evidence for a mechanism involving cleavage of the anomeric bond and formation of 2-
acetamidoglucal and UDP as intermediates.
Additional studies with alternative substrates were used to gain more information about
the mechanism of the epimerase. A 3'-deoxy analog (3'-deoxy-UDP-GlcNAc) was prepared
and incubated with the epimerase to test for mechanisms involving the formation of a keto-intermediate
at this center. The absence of epimerization with this substrate of the epimerase
failed to rule out any such mechanisms, but suggested the need of the 3"-hydroxyl group as a
necessary element for the recognition of the substrate. A trifluoroacetamido analog (UDP-GlCNAC-
F3) was also prepared and proved to be an alternative substrate of the enzyme with a
kcat of 2.6 x 10-2 s-1 an apparent Km of 3.40 ± 0.37 mM and a Hill coefficient of 1.6 ± 0.2. This
compound was also an acceptable substrate for two sugar transferases.
The possible involvement of neighboring group participation of the acetamido group
during the epimerization was also tested by incubation of the oxazoline of ManNAc with the
epimerase under conditions that permitted the detection of enzyme catalyzed formation of 2-
acetamidoglucal or sugar nucleotides from this putative intermediate. The negative result
obtained in this experiment along with the behavior of the epimerase with the
trifluoroacetamido analog suggests (but does not prove) that neighboring group participation is
not involved in the epimerization.
The minimal description of the mechanism of the epimerase is unprecedented and
involves the anti elimination of UDP from UDP-GlcNAc (syn from UDP-ManNAc) to form 2-
acetamidoglucal and UDP as reaction intermediates followed by a syn addition (anti if started
from UDP-ManNAc) of UDP to the double bond of the glycal intermediate to generate the
sugar nucleotide epimer. Precedents for this process as well as a more detailed discussion of
the type of elimination involved and future directions are also presented. [Scientific formulae used in this abstract could not be reproduced.]
|
|---|
