Probing the Roles of Active Site Residues in D-Xylose Isomerase

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 -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 D-Xylose isomerase (EC 5.3.1.5) catalyzes the reversible interconversions of D-xylose to D-xylulose and D-glucose to Dfructose. The D-xylose isomerase gene (xylA) has been cloned from a variety of bacterial sources (1-12) and structures of the Streptomyces rubiginosus (13-15), Arthrobacter (16 -18), Actinoplanes missouriensis (19), and Streptomyces olivochromogenes (20, 21) enzymes have been determined by x-ray crystallography. The catalytic domains fold as eight-stranded / barrel motifs and contain a well conserved active site with two divalent metal ions (17). All D-xylose isomerases require Mg 2 , Co 2 , or Mn 2 ions for activity, suggesting that they have