The role of active-site aromatic and polar residues in catalysis and substrate discrimination by xylose isomerase.
The functions of individual amino acid residues in the active site of Thermoanaerobacterium thermosulfurigenes D-xylose ketol-isomerase (EC 5.3.1.5) were studied by site-directed substitution. The role of aromatic residues in the active-site pocket was not limited to the creation of a hydrophobic environment. For example, Trp-188 provided for substrate binding and Trp-139 allowed for the discrimination between D-xylose and D-glucose. Substrate discrimination was accomplished by steric hindrance caused by the side chain of Trp-139 toward the larger glucose molecule. Preference of the enzyme for the alpha-anomer of glucose depended on the His-101/Asp-104 pair. Wide differences observed in the catalytic constant (kcat) for alpha- versus beta-glucose in the wild-type enzyme and the fact that only the kcat for alpha-glucose was changed in the His-101-->Asn mutants strongly suggest that the substrate molecule entering the hydride-shift step is still in the cyclic form. On the basis of these results a revised hypothesis for the catalytic mechanism of D-xylose isomerase has been proposed that involves His-101, Asp-104, and Asp-339 functioning as a catalytic triad.
Department of Microbiology, Michigan State University, East Lansing 48824.
M
M
Meng
M
M
Bagdasarian
J G
JG
Zeikus
D019747:Aldose-Ketose Isomerases; D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity; D014994:Xylose / Q000378:metabolism
D014994:Xylose; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D005947:Glucose