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The possible roles of four histidine residues in the active site of yeast transketolase were examined by site-directed mutagenesis. Replacement of the invariant His69 with alanine yielded a mutant enzyme with 1.5% of the specific activity of the wild-type enzyme and with an increased KM for the donor. This residue is located at the bottom of the substrate cleft close to the C1 hydroxyl group of the donor substrate, and the side chain of His69 might be required for recognition of this hydroxyl group and possibly for maintenance of the proper orientation of the reaction intermediate, (alpha, beta-dihydroxyethyl)thiamin diphosphate. Amino acid replacements of His481 by alanine, serine, and glutamine resulted in mutant enzymes with significantly increased KM values for the donor substrate and specific activities of 4.4%, 1.9%, and 5.5% of the wild-type enzyme. The kinetic data suggest that this residue, although close to the C2 carbonyl oxygen of the substrate, is not absolutely required for stabilization of the negative charge that develops at this oxygen in the transition state. This points toward the 4'-NH2 group of the pyrimidine ring of thiamin diphosphate as the major source of charge stabilization. Mutations at positions His30 and His263 result in mutant enzymes severely impaired in catalytic activity (1.5% and less of the activity of wild-type transketolase). The KM value for the donor substrate was increased for the His30Ala mutant but remained unchanged in the His263Ala enzyme. The side chains of both residues interact with the C3 hydroxyl group of the donor substrate, and the results indicate that the two residues act in concert during proton abstraction of the C3 hydroxyl proton during catalysis.

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