UDP-(5F)-GlcNAc acts as a slow-binding inhibitor of MshA, a retaining glycosyltransferase

Abstract

Glycosyltransferase enzymes play important roles in numerous cellular pathways. Despite their participation in many therapeutically relevant pathways, there is a paucity of information on how to effectively inhibit this class of enzymes. Here we report that UDP-(5F)-GlcNAc acts as a slow-binding, competitive inhibitor of the retaining glycosyltransferase MshA from Corynebacterium glutamicum (K(i) approximately 1.6 muM). The kinetic data are consistent with a single-step inhibition mechanism whose equilibration is slow relative to catalysis. We believe that this is the first slow-onset inhibitor to be reported for the glycosyltransferase family of enzymes. The potent inhibition of the enzyme by the fluoro-substituted substrate is consistent with the involvement of an oxocarbenium transition-state structure, which has been previously proposed for this family of enzymes. Additionally, although several members of the GT-B enzyme family, including MshA, have been shown to undergo a conformational change upon UDP-GlcNAc binding, the kinetic data are inconsistent with a two-step inhibition mechanism. This suggests that there may be other conformations of the enzyme that are useful for the design of inhibitors against the large family of GT-B glycosyltransferase enzymes.

Figure 1

(A) Kinetics of time-dependent inhibition of CgMshA by UDP-(5F)-GlcNAc. Progress curves for MshA in the absence (blue) and presence (black) of UDP-(5F)-GlcNAc. The red lines are a fit to eq 1. Saturating substrate concentrations of 2 mM UDP-GlcNAc and 2 mM L-I1P were used in this experiment. (B) Effect of pre-incubation with UDP-(5F)-GlcNAc on CgMshA activity initiated with UDP-GlcNAc. A solution of 8 nM enzyme, 2 mM L-I1P, and 5 µM UDP-(5F)-GlcNAc was incubated at 25 °C for 10 minutes prior to the addition of 1 mM UDP-GlcNAc (black line). The volume of UDP-GlcNAc added was 1% of the reaction volume. The blue line represents the same experiment in the absence of UDP-(5F)-GlcNAc. See Supporting Information for full experimental details.

Figure 2

Plot of k_obs versus [UDP-(5F)-GlcNAc]. The values for k_obs were determined by fits of the progress curves to equation 1. The solid black line is a fit of the data to equation 2.

Scheme 1

Scheme 2

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