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 aroma tic residues in the active-site pocket was not limited to the creation of a hydrophobic environment. For example, Trp-188 provided for subst rate binding and Trp-139 allowed for the discrimination between D-xylo se and D-glucose. Substrate discrimination was accomplished by steric hindrance caused by the side chain of Trp-139 toward the larger glucos e molecule. Preference of the enzyme for the alpha-anomer of glucose d epended on the His101/Asp-104 pair. Wide differences observed in the c atalytic constant (k(cat)) for alpha- versus beta-glucose in the wild- type enzyme and the fact that only the k(cat) for alpha-glucose was ch anged in the His-101 --> Asn mutants strongly suggest that the substra te molecule entering the hydride-shift step is still in the cyclic for m. On the basis of these results a revised hypothesis for the catalyti c mechanism of D-xylose isomerase has been proposed that involves His- 101, Asp-104, and Asp-339 functioning as a catalytic triad.