Probing the roles of active site residues in D-xylose isomerase.
The roles of active site residues His54, Phe94, Lys183, and His220 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 His54 suggests that His54 does not catalyze ring-opening as a general acid. His54 appears to be involved in anomeric selection and stabilization of the acyclic transition state by hydrogen bonding. Phe94 stabilizes the acyclic-extended transition state directly by hydrophobic interactions and/or indirectly by interactions with Trp137 and Phe26. Lys183 and His220 mutants have little or no activity and the structures of these mutants with D-xylose reveal cyclic alpha-D-xylopyranose. Lys183 functions structurally by maintaining the position of Pro187 and Glu186 and catalytically by interacting with acyclic-extended sugars. His220 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.
Whitaker
R D
RD
Department of Food Science, Cornell University, Ithaca, New York 14853, USA.
Cho
Y
Y
Cha
J
J
Carrell
H L
HL
Glusker
J P
JP
Karplus
P A
PA
Batt
C A
CA
D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004926:Escherichia coli; D006639:Histidine; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D014364:Tryptophan
D017931:DNA Primers; D011994:Recombinant Proteins; D010649:Phenylalanine; D006639:Histidine; D014364:Tryptophan; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D008239:Lysine