Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase.
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.
Contestabile
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Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Università La Sapienza, Roma, Italy.
Angelaccio
S
S
Bossa
F
F
Wright
H T
HT
Scarsdale
N
N
Kazanina
G
G
Schirch
V
V
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*
D014443:Tyrosine; D012696:Glycine Hydroxymethyltransferase