Purification of 5,10-Methylenetetrahydrofolate Dehydrogenase from Salmonella typhimurium and Its Inhibition by Purine Nucleotides

Abstract 5,10-Methylenetetrahydrofolate dehydrogenase has been purified nearly 200-fold from Salmonella typhimurium. The purified enzyme is highly unstable and can only be preserved as a suspension in ammonium sulfate at 50% saturation. The apparent Km values obtained for d,l-methylenetetrahydrofolate and NADP+ were 9.2 x 10-5 m and 4.5 x 10-5 m, respectively. The enzyme is inhibited by purine nucleotides. ATP acts as a noncompetitive inhibitor with respect to d,l-methylenetetrahydrofolate, but there is some competition between ATP and NADP+. Inhibition is incomplete even at high ATP concentrations, nearly 20% of activity being retained at ATP levels more than 10 times the apparent Ki concentration. The partial desensitization of the enzyme to inhibition was obtained by variations in pH and by p-hydroxymercuribenzoate. The inhibition by purine nucleotides could serve as a physiologically important control mechanism preventing the unnecessary accumulation of the 5,10-methenyl and 10-formyl derivatives of tetrahydrofolate, both of which are involved in the transformylation reactions of purine biosynthesis, and the retention of activity at high inhibitor concentrations may be essential in order to maintain enough formyl-tetrahydrofolate for transformylations other than those involved in purine synthesis.