DESIGN, SYNTHESIS, AND CHARACTERIZATION OF A POTENT XYLOSE ISOMERASE INHIBITOR, D-THREONOHYDROXAMIC ACID, AND HIGH-RESOLUTION X-RAY CRYSTALLOGRAPHIC STRUCTURE OF THE ENZYME-INHIBITOR COMPLEX

The binding of a potent inhibitor to the enzyme D-xylose isomerase from Streptomyces olivochromogenes was examined by kinetics and X-ray crystallography. The inhibitor D-threonohydroxamic acid (THA) was designed to mimic the putative transition state of the isomerization step catalyzed by the enzyme on the substrate xylose. THA was synthesized and found to be a slow-binding competitive inhibitor with the substrate glucose. The K-i less than or equal to 100 nM was at least one million-fold less than the K-M for glucose. The X-ray crystallographic structure of xylose isomerase with THA soaked into the crystals (concentration = 1000K(i)) was obtained to 1.6-Angstrom resolution and refined to an R factor of 21.6%. The free enzyme and the enzyme in the xylose isomerase-THA complex show no significant structural differences. THA binds in an analogous fashion to glucose, in a linear conformation, forming ligands with Mg-1 and Mg-2 and hydrogen bonds with His53 and Lys182. On the basis of these similarities to glucose binding and its potent inhibition, we propose that THA resembles the transition state for the enzyme-catalyzed hydride transfer reaction. The THA C2 hydroxyl forms a bridging ligand between Mg-1 and Mg-2; it must be deprotonated to do so. By analogy, we propose that, during the catalytic reaction, C2 of the substrate glucose is deprotonated, and that this proton can be moved to the C1 hydroxyl concomitant with hydride transfer. We find evidence for metal movement during catalysis upon deprotonation of the C2 hydroxyl, to allow formation of a bridging ligand. Ln addition, the observation in the xylose isomerase-THA complex of a water molecule bound to Mg-2 instead of a hydroxide (as seen in the native structure) suggests that protonation of the hydroxyl occurs in a step following ring opening. The metal-bound hydroxide ion may act as a general base to deprotonate the C2 hydroxyl of the substrate. Due to their ability to act as metal ligands, hydroxamic acids may be general inhibitors of dimetallic enzymes.