The reaction of tetrahydropteroylglutamic acid and related hydropteridines with formaldehyde

Abstract

4-Hy-droxy-6-methyltetrahydropteridine has been synthesized and shown to bind formaldehyde in dilute solution. The evidence indicates a reversible reaction, the dissociation constant for the product being about 0.03. 2-Amino-4-hydroxy-6-methyltetrahydropteridine and tetrahydro-quinoxaline react similarly with formaldehyde, the corresponding dissociation constants being 0.04 and 0.01 respectively. It is concluded that such hydropteridines undergo a reversible reaction with formaldehyde to form N5-hydroxymethyl derivatives. This conclusion is strengthened by the fact that there is little binding of formaldehyde by N5-formyl-4-hydroxy-6-methyltetrahydropteridine. With 4-hydroxy-6-methyltetrahydropteridine, equilibrium is reached in a few minutes at room temperature when the initial concentration of pteridine is 4 m[image] and the initial concentration of formaldehyde is 0.5 m[image]. Tetrahydrop-teroylglutamic acid (THGA) binds 0.7-0.95 molecular proportion of formaldehyde, corresponding to the formation of a product with a dissociation constant of 4 x 10-4 or lower, the reaction being completed in 5-7 minutes when the reactants have an initial concentration of 0.4 m[image]. There is no detectable binding of formaldehyde by N5-formyl-THGA, but the dissociation constants for the compounds formed by N10-formyl-THGA and dihydropteroylglutamic acid were found to be 0.01. A new procedure has been developed for preparation of serine trans-hydroxymethylase. The more highly purified enzyme obtained by this method has been used to re-determine the Michaelis constant for THGA. Dihydropteroylglutamic acid and N10-formyl-THGA did not activate the enzyme. The non-enzymic combination of formaldehyde and THGA proceeded so rapidly that its rate greatly exceeded the maximum rate of enzymic serine synthesis obtained under the same conditions in vitro. It may therefore be assumed that non-enzymic combination of formaldehyde and THGA is the first step in serine synthesis. Serine trans-hydroxymethylase preparations did not accelerate combination of formaldehyde and THGA. It is concluded that THGA and formaldehyde react reversibly, possibly forming N5N10-methylenetetrahydro-pteroylglutamic acid. In view of the weakness with which N-10 of THGA binds formaldehyde, N5-hydroxymethyltetrahydropteroylglutamic acid may be an intermediate in the formation and dissociation of N5N10-methylenetetrahydropteroylglutamic acid.