title: Properties of a serine hydroxymethyltransferase in which an active site histidine has been changed to an asparagine by site-directed mutagenesis.
abstract: Histidine 228 at the active site of Escherichia coli serine hydroxymethyltransferase was replaced with an asparagine. The mutant enzyme was expressed in a strain of E. coli that lacks wild type enzyme. Absorption spectra, circular dichroism spectra, and differential scanning calorimetry thermograms suggest that the amino acid change at the active site causes no detectable change in the tertiary structure of the enzyme. Kinetic studies demonstrated that kcat for the mutant enzyme is about 25% of the value for the wild type enzyme with either L-serine or allothreonine as substrate. Km or Kd values for amino acid substrates and reduced folate compounds were 2-10-fold larger with the mutant enzyme. The rate of interconversion of several enzyme-glycine complexes showed that the conversion of the external aldimine to the quinoid complex is not the rate-determining step for either the mutant or wild type enzyme in the presence of tetrahydrofolate. The binding of L-serine to the wild type enzyme gives a more thermally stable enzyme and increases its affinity for tetrahydrofolate. These effects are not found when L-serine binds to the mutant enzyme. The studies demonstrate that histidine 228 is not a catalytically essential residue and suggest that it is involved in interacting with either the amino acid substrate or the enzyme-bound pyridoxal phosphate.
mesh_terms: D001216:Asparagine / Q000378:metabolism*; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D012696:Glycine Hydroxymethyltransferase / Q000235:genetics* / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D013329:Structure-Activity Relationship; D014166:Transferases / Q000235:genetics*
chemical_list: D006639:Histidine; D001216:Asparagine; D014166:Transferases; D012696:Glycine Hydroxymethyltransferase