4-CHLOROTHREONINE IS SUBSTRATE, MECHANISTIC PROBE, AND MECHANISM-BASED INACTIVATOR OF SERINE HYDROXYMETHYLTRANSFERASE

Serine hydroxymethyltransferase catalyzes the cleavage of a variety of beta-hydroxy-L-amino acids to form glycine and aldehyde products. 4-chloro-L-threonine has been synthesized and shown to be both a substrate and a mechanism-based inactivator of serine hydroxymethyltransferase. k(cat) values for the formation of glycine in the absence of tetrahydrofolate were determined for 4-chloro-L-threonine and other beta-hydroxyamino acid substrates; an inverse relationship between the rate of cleavage of the amino acid and the electrophilicity of the product aldehyde was demonstrated. 4-Chloro-L-threonine inactivates serine hydroxymethyltransferase in a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations. Our evidence suggests that 4-chlorothreonine undergoes aldol cleavage, and generation of chloroacetaldehyde at the active site of the enzyme results in inactivation. Serine or glycine protect the enzyme against inactivation by chlorothreonine, while tetrahydrofolate does not. The enzyme is also protected from inactivation by 2-mercaptoethanol or by alcohol dehydrogenase and NADH. These studies suggest that halothreonine derivatives that generate electrophilic aldehyde products will be effective inhibitors of serine hydroxymethyltransferase and might be potentially useful chemotherapeutic agents.