| Abstract
| - Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shownthat Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex ofEscherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxylof Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate groupof the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65.The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphouswith the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of thismutant enzyme in catalyzing reactions with serine, glycine, allothreonine, d- and l-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with d- and l-alanine,glycine in the absence of tetrahydrofolate, and 5,10-methenyltetrahydrofolate were not significantly changed.However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solventα-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity forthese reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65Fmutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These dataare consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to anopen structure.
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