title: Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase.
abstract: Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase 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 hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 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 isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant 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 solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Università La Sapienza, Roma, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Contestabile
authors.forename: S
authors.initials: S
authors.lastname: Angelaccio
authors.forename: F
authors.initials: F
authors.lastname: Bossa
authors.forename: H T
authors.initials: HT
authors.lastname: Wright
authors.forename: N
authors.initials: N
authors.lastname: Scarsdale
authors.forename: G
authors.initials: G
authors.lastname: Kazanina
authors.forename: V
authors.initials: V
authors.lastname: Schirch
mesh_terms: D000818:Animals; D002384:Catalysis; D018360:Crystallography, X-Ray; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation; D011817:Rabbits; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D014443:Tyrosine; D012696:Glycine Hydroxymethyltransferase