Mechanism for the coupling of ATP hydrolysis to the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate.
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.
Huang
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T
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298, USA.
Schirch
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V
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D005574:Formate-Tetrahydrofolate Ligase / Q000378:metabolism*; D007700:Kinetics; D002955:Leucovorin / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D011817:Rabbits; D013237:Stereoisomerism; D013763:Tetrahydrofolates / Q000378:metabolism*
D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D000255:Adenosine Triphosphate; D005574:Formate-Tetrahydrofolate Ligase; D002955:Leucovorin