Probing the roles of active site residues in D-xylose isomerase

Abstract

The roles of active site residues His(54), Phe(94), Lys(183), and His(220) in the Streptomyces rubiginosus D-xylose isomerase were probed by site-directed mutagenesis. The kinetic properties and crystal structures of the mutant enzymes were characterized. The pH dependence of diethylpyrocarbonate modification of His(54) suggests that His(54) does not catalyze ring-opening as a general acid. His(54) appears to be involved in anomeric selection and stabilization of the acyclic transition state by hydrogen bonding. Phe(94) stabilizes the acyclic-extended transition state directly by hydrophobic interactions and/or indirectly by interactions with Trp(137) and Phe(26). Lys(183) and His(220) mutants have little or no activity and the structures of these mutants with D-xylose reveal cyclic alpha-D-xylopyranose. Lys(183) functions structurally by maintaining the position of Pro(187) and Glu(186) and catalytically by interacting with acyclic-extended sugars. His(220) provides structure for the M2-metal binding site with properties which are necessary for extension and isomerization of the substrate. A second M2 metal binding site (M2') is observed at a relatively lower occupancy when substrate is added consistent with the hypothesis that the metal moves as the hydride is shifted on the extended substrate.