Mechanistic investigation of UDP-galactopyranose mutase from Escherichia coli using 2-and 3-fluorinated UDP-galactofuranose as probes

The galactofuranose moiety found in many surface constituents of microorgan isms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring con traction reaction catalyzed by UDP-Galp mutase. This enzyme, which has been isolated from several bacterial sources, is a flavoprotein. To study this catalysis, the cloned Escherichia coil mutase was purified and two fluorina ted analogues, UDP-[2-F]Galf(9) and UDP-[3-F]Galf(10), were chemically synt hesized. These two compounds were found to be substrates for the reduced UD P-Galp mutase with the K-m values determined to be 65 and 861 muM for 9 and 10, respectively, and the corresponding k(cat) values estimated to be 0.03 3 and 5.7 s(-1). Since the fluorine substituent is redox inert, a mechanism initiated by the oxidation of 2-OH or 3-OH on the galactose moiety can thu s be firmly ruled out. Furthermore, both 9 and 10 are poorer substrates tha n UDP-Galf, and the rate reduction for 9 is especially significant. This fi nding may be ascribed to the inductive effect of the 2-F substituent that i s immediately adjacent to the anomeric center, and is consistent with a mec hanism involving formation of oxocarbenium intermediates or transition stat es during turnover. Interestingly, under nonreducing conditions, compounds 9 and 10 are not substrates, but instead are inhibitors for the mutase. The inactivation by 10 is time-dependent, active-site-directed. and irreversib le with a K-I of 270 muM and a k(inact) of 0.19 min(-1). Since the K-I valu e is similar to K-m, the observed inactivation is unlikely a result of tigh t binding. To our surprise, the inactivated enzyme could be regenerated in the presence of dithionite, and the reduced enzyme is resistant to inactiva tion by these fluorinated analogues. It is possible that reduction of the e nzyme-bound FAD may induce a conformational change that facilitates the bre akdown of the putative covalent enzyme-inhibitor adduct to reactivate the e nzyme. It is also conceivable that the reduced flavin bears a higher electr on density at N-1, which may play a role in preventing the formation of the covalent adduct or facilitating its breakdown by charge stabilization of t he oxocarbenium intermediates/transition states. Clearly, this study has le d to the identification of a potent inactivator (10) for this enzyme, and s tudy of its inactivation has also shed light on the possible mechanism of t his mutase.