Mechanistic insight into serine hydroxymethyltransferase through the use of substrate analogs.

Abstract

Serine hydroxymethyltransferase catalyzes the reversible transfer of the $\beta$-carbon from serine to tetrahydrofolate to form glycine and methylenetetrahydrofolate. Serine hydroxymethyltransferase plays the major role in charging tetrahydrofolate with one-carbon units, an essential step in the biosynthesis of the purines and thymidylate required for DNA synthesis. The goals of this work were as follows: first, synthesis of the serine analog 4-chlorothreonine; second, testing of 4-chlorothreonine as a substrate both in the presence and absence of tetrahydrofolate; third, testing of 4-chlorothreonine as a mechanism-based inhibitor; and finally, testing the effects of the tetrahydrofolate analog, 5,10 dideazatetrahydrofolate. 4-Chlorothreonine was synthesized and tested for its ability to act as a substrate for serine hydroxymethyltransferase, as were a number of other $\beta$-hydroxyamino acids. The rate of glycine formation in the absence of tetrahydrofolate increases as the alkyl or aryl substituents on the $\beta$-carbon become more electron-donating. A linear free energy relationship was found to exist between the rate of cleavage of $\beta$-hydroxyamino acids and the hydration equilibrium of the product aldehyde. Stimulation of the rate of cleavage of $\beta$-hydroxyamino acids by tetrahydrofolate has previously only been observed with those substrates generating the extremely electrophilic aldehyde, formaldehyde. The serine hydroxymethyltransferase-catalyzed cleavage of 4-chlorothreonine is stimulated four-fold by tetrahydrofolate, but not by the non-nucleophilic 5,10-dideazatetrahydrofolate. These observations are discussed in terms of proposed mechanisms for serine hydroxymethyltransferase. The proposed mechanism of inactivation of serine hydroxymethyltransferase by 4-chlorothreonine involves aldol cleavage of the C$\sb\alpha$-C$\sb\beta$ bond and release of chloroacetaldehyde. The rate of inactivation is time- and concentration-dependent, showing saturation at high concentrations of 4-chlorothreonine. Both serine and glycine, protect the enzyme from inactivation by 4-chlorothreonine. The enzyme is also protected from inactivation by $\beta$-mercaptoethanol and alcohol dehydrogenase. The above results indicate that 4-chlorothreonine is a mechanism-based inhibitor and suggest that inactivation occurs after release of chloroacetaldehyde from the active site.