Probing the roles of active site residues in D-xylose isomerase
| Autor: | Jaeho Cha, J. P. Glusker, Yunje Cho, H. L. Carrell, Richard D. Whitaker, P.A. Karplus, Carl A. Batt |
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| Rok vydání: | 1995 |
| Předmět: |
Xylose isomerase
Models Molecular Stereochemistry Protein Conformation Phenylalanine Molecular Sequence Data Metal Binding Site Isomerase Crystallography X-Ray Biochemistry Polymerase Chain Reaction Protein structure Escherichia coli Point Mutation Histidine Amino Acid Sequence Binding site Cloning Molecular Molecular Biology Aldose-Ketose Isomerases DNA Primers Binding Sites biology Base Sequence Chemistry Lysine Tryptophan Active site Substrate (chemistry) Cell Biology Recombinant Proteins Streptomyces Kinetics Streptomyces rubiginosus biology.protein Mutagenesis Site-Directed Carbohydrate Epimerases |
| Zdroj: | The Journal of biological chemistry. 270(39) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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