Mechanistic investigations of unsaturated glucuronyl hydrolase from Clostridium perfringens

Abstract

Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct (1)H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2.

Keywords: Carbohydrate Metabolism; Chemical Biology; Enzyme; Enzyme Mechanisms; Glycosaminoglycan; Glycosidases; Glycoside Hydrolases; Hydratases; Isotope Effects.

FIGURE 1.

Currently accepted mechanism for hydrolysis by UGL, initiated by direct hydration of the carbon 4 to carbon 5 double bond.

FIGURE 2.

¹H NMR monitoring of ΔGlcUA glycoside hydrolysis by a high concentration of UGL. A, 3-nitrophenyl ΔGlcUA; B, thiophenyl ΔGlcUA. A water suppression pulse program was used for automated variable delay recording of spectra for the 1st h, followed by manual recording of later spectra using a standard proton pulse program with updated tuning and shimming. The overall reaction and main products are as shown top left, with main peaks labeled in the top spectrum (products) and lowest spectrum (starting material). The H-1 peak label for the products is adjacent to the relevant peak, which overlaps substantially with the HOD residual solvent peak and so is also suppressed in the first product spectrum.

FIGURE 3.

Anticipated reaction scheme for inactivation of UGL by 1-fluoro-ΔGlcUA fluoride. Formation of the highly reactive acyl fluoride moiety in the enzyme active site would lead to nonspecific derivatization of nucleophilic amino acid side chains close to the active site, some of which will inactivate the enzyme.

FIGURE 4.

Time-dependent inactivation of UGL by 1-fluoro-ΔGlcUA fluoride. Concentrations of potential inactivator are 0 (■), 5.5 (▵), 11 (♦), 22.1 (□), 36.8 (●), and 51.5 (×) mm. Fits are to first order decay.

FIGURE 5.

A, modified mechanism for the hydration reaction of UGL, proceeding through a covalent glycosyl-enzyme intermediate. B, modified mechanism for the hydration reaction of UGL, proceeding through a non-enzyme linked epoxide intermediate.

FIGURE 6.

Precedent for aspects of the mechanisms in Fig. 5 from nonenzymatic carbohydrate chemistry. A, precedent for pyranose ring opening; B, precedent for epoxide intermediate formation. C and D, precedent for intramolecular epoxide opening by a carbonyl group. See text for references.

FIGURE 7.

Anticipated mechanism for trapping of UGL by 5-fluoro product analogs and structures of the analogs investigated.

FIGURE 8.

Attempted time-dependent inactivation of UGL by 2,3-difluoro-Kdn at 0 (▵), 0. 2 (■), 2 (□), and 20 (×) mm (A) and 4-deoxy-1,5-difluoro-iduronic acid at 0 (×) and 5 (●) m mm (B).