Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase

Crystal structures of human and rabbit cytosolic serine hydroxymethyltransf erase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymet hyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of T yr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of 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 isomorpho us with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with seri ne, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofo late substrates were determined. The properties of the enzyme with D- and L -alanine, glycine in the absence of tetrahydrofolate, and 5,10-methenyltetr ahydrofolate were not significantly changed. However, catalytic activity wa s greatly decreased for serine and allothreonine cleavage and for the solve nt ct-proton exchange of glycine in the presence of tetrahydrofolate. The d ecreased catalytic activity for these reactions could be explained by a gre ater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimi ne. These data are consistent with a role for the Tyr65 hydroxyl group in t he conversion of a closed active site to an open structure.