Serine Transhydroxymethylase

Abstract

Abstract A number of substrate analogues of serine transhydroxymethylase were tested as competitive inhibitors and substrates. Our previous results show that l-serine, l-threonine, allothreonine, and d-alanine serve as substrates. In this study, we found that threo- and erythro-β-phenylserine were cleaved to benzaldehyde and glycine. Tetrahydrofolate was not required for this reaction. Substrate analogues which contained an amino group adjacent to a carboxyl, phosphonate, or sulfonate group were bound by the enzyme. Analogues which lacked either the amino group or a carboxyl group of the amino acid had little if any affinity for the active site. The only exceptions to this were several sulfhydryl compounds. Several d-amino acids were competitive inhibitors with d-cysteine being the most effective. This shows that the configuration around the α-carbon of the amino acid is not critical for substrate binding. The variation of Vmax and Km with pH for the substrate allothreonine was determined. Both Vmax and Km were found not to change from pH 5.8 to 7.6. However, an anion binding site on the enzyme with a pK of 6.2 was found. This site showed little specificity for anions with all anions tested serving as competitive inhibitors. The enzyme-glycine complex exhibits absorption maxima at 343, 425, and 495 mµ. The equilibrium between the complexes absorbing at 343 and 425 mµ was found to be sensitive to both temperature and pH. A group on the enzyme-glycine complex with a pK of 6.9 was found to be involved. The temperature sensitivity was observed to be due in part to a large increase in entropy, which suggests that conformational changes in the protein are also involved in the interconversion of the enzyme-glycine complexes.