Abstract
5,10-Methenyltetrahydrofolate synthetase catalyzes the irreversible conversion of 5-formyl-tetrahydropteroylpolyglutamates (5-CHO-H4PteGlu(n)) to 5,10-methenyltetrahydropteroylpolyglutamates (5, 10-CH(+)-H4PteGlu(n)). The equilibrium of the nonenzymatic reaction, which equilibrates slowly in the absence of enzyme, greatly favors 5-CHO-H4PteGlu(n). The enzyme couples the reaction to the hydrolysis of ATP shifting the equilibrium to favor 5,10-CH(+)-H4PteGlu(n). Substrate-dependent non-equilibrium isotope exchange of [3H]ADP into ATP was observed, suggesting the formation of a phosphorylated intermediate of 5-CHO-H4PteGlu(n) during the enzyme-catalyzed reaction. The competitive inhibitor 5-formyltetrahydrohomofolate also supported the ADP to ATP exchange, suggesting that this molecule could also form a phosphorylated intermediate. The initial rates of the ADP-ATP exchange with saturating ADP were about 70 s-1 for both compounds, while the kcat values for product formation were 5 s-1 for 5-CHO-H4PteGlu(n) and 0.005 s-1 for 5-formyltetrahydrohomofolate. Starting with 5(-)[18O]CHO-H4PteGlu(n), it was shown by 31P NMR that the formyl oxygen of the substrate was transferred to the product phosphate during the reaction. This further supports the existence of a phosphorylated intermediate. The formyl group of 5-CHO-H4PteGlu(n) is known to be an equilibrium mixture of two rotamers. Stopped-flow analysis of the enzymatic reaction showed that only one of the rotamers serves as a substrate for the enzyme.
Highlights
5,10-Methenyltetrahydrofolate synthetase (MS)1 (EC 6.3.3.2), referred to as 5-formyltetrahydrofolate cyclodehydrase, catalyzes the ATP-dependent conversion of 5-formyltetrahydrofolate (5-CHO-H4PteGlun) to 5,10-methenyltetrahydrofolate (5,10-CHϩ-H4PteGlun) (Reaction I)
Because 5-CHO-H4PteGlun is the only tetrahydrofolate derivative that is stable to oxidative degradation, these studies suggested that a physiological role is to serve as a storage form of this coenzyme in dormant stages of cellular life cycles
For 5-CHO-H4PteGlu to be effective as a rescue agent and an enhancer of 5-fluorouracil toxicity, it must first be converted to 5,10-CHϩ-H4PteGlu through MS activity
Summary
The origin and function of 5-CHO-H4PteGlun has been a source of confusion and debate [8]. Neither an enzymatic source nor function of 5-CHO-H4PteGlun was known This suggested that the function of MS was to serve as a salvage pathway for the reincorporation of this nonenzymatically produced folate derivative into the one-carbon pool. Making Reaction I irreversible would be beneficial to a cell whether 5-CHO-H4PteGlun is formed nonenzymatically, is used as a storage form for folates, or functions in regulation by inhibiting enzymes in one-carbon metabolism. It has been shown that doubling intracellular concentrations of 5-CHOH4PteGlun, by inhibiting MS activity, suppressed growth of MCF-7 human breast cells by 80% [13] This suggests that MS could be a potentially important enzyme as a target in chemotherapy. By using NMR spectroscopy and isotope exchange techniques, a 5-CHO-H4PteGlun phosphorylated intermediate was demonstrated The role of this intermediate in making Reaction I irreversible is discussed
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