Folylpolyglutamates as substrates and inhibitors of folate-dependent enzymes

Abstract

The true intracellular substrates for folate-dependent enzymes are folylpolyglutamates. We have used measurements of the Ki values of folylpolyglutamate dead end inhibitors to assess the relative affinities of folate-dependent enzymes for folate derivatives of different polyglutamate chain lengths. Studies of four enzymes from pig liver, methylenetetrahydrofolate reductase, serine hydroxymethyltransferase, methylenetetrahydrofolate dehydrogenase and thymidylate synthase, have indicated that folylpolyglutamate inhibitors are bound 3-500 fold more tightly than the corresponding monoglutamates. The individual enzymes differ in their selectivity for polyglutamate vs. monoglutamate inhibitors, and in the chain length associated with the greatest affinity of enzyme for inhibitor. We have also examined the effect of polyglutamate chain length on the catalytic parameters associated with folate substrates. Two enzymes, methylenetetra-hydrofolate reductase and serine hydroxymethyltransferase, show decreases in Km values for folypolyglutamate substrates. Methylenetetrahydrofolate dehydrogenase shows no detectable differences in the catalytic parameters of polyglutamate vs. monoglutamate substrates and no change in the order of substrate addition or product release. Thymidylate synthase shows small effects of Km and Vmax values, but the order of addition of substrates and of release of products is reversed with polyglutamate as compared with monoglutamate substrates. Our studies with thymidylate synthase from L. casei have shown that the bacterial enzyme also exhibits a greatly increased affinity for polyglutamate vs. monoglutamate derivatives of folic acid, and that reversal in the order of substrate addition and product release also occurs with polyglutamate as compared with monoglutamate substrates. We have also studied the polyglutamate specificity of methionine synthase, which is responsible for the conversion of CH3---H4PteGlu1 into H4PteGlu1. This reaction is required for the incorporation of plasma folate into the cellular folate pool, because methyltetrahydrofolate is a poor substrate for folylpolyglutamate synthetase. Our studies demonstrate that CH3---H4PteGlu1 metabolism is potently inhibited in the presence of CH3---H4PteGlu6, and suggest that incorporation of plasma CH3---H4PteGlu1 will only occur when methylenetetrahydrofolate reductase is inhibited by adenosylmethionine and cellular pools of CH3---H4PteGlu6 are at very low levels.