The roles of active site residues His, Phe, Lys, and His in the Streptomyces rubiginosusD-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 suggests that His does not catalyze ring-opening as a general acid. His appears to be involved in anomeric selection and stabilization of the acyclic transition state by hydrogen bonding. Phe stabilizes the acyclic-extended transition state directly by hydrophobic interactions and/or indirectly by interactions with Trp and Phe. Lys and His mutants have little or no activity and the structures of these mutants with D-xylose reveal cyclic -D-xylopyranose. Lys functions structurally by maintaining the position of Pro and Glu and catalytically by interacting with acyclic-extended sugars. His 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

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