Calorimetric Studies of Thymidylate Synthesis

Abstract

Abstract Heats of reaction for dihydrofolate (DHF) reductase (Reaction 1), for the reaction of formaldehyde with tetrahydrofolate (THF) (Reaction 2), and for thymidylate (dTMP) synthetase (Reaction 3) were measured in an isothermal conduction calorimeter operated at 25 ± 1° employing enzymes for Reactions 1 and 3 isolated from Lactobacillus casei. NADPH + DHF + H+ ⇄ NADP+ + THF ΔH1 = -12.6 ± 0.3 Cal/mole (1) CH2(OH) + THF ⇄ methylene-THF (CH2 - THF) + 2H2O ΔH2 = -25.0 ± 5 Cal/mole (2) CH2 - THF + dUMP ⇄ DHF + dTMP ΔH3 = -9.5 ± 1.3 Cal/mole (3) Reaction 2 occurs without enzyme and is accompanied by a large decrease in entropy. The observed enthalpy values in combination with the appropriate equilibrium constants from the literature indicate that the experimentally observed irreversibility of Reaction 3 is due in part to a large positive entropy change. The sum of Reactions 1, 2, and 3 gives the over-all Reaction 4 in which the reduced folates act catalytically. NADPH + H+ + CH2(OH)2 + dUMP ⇄ NADP+ + dTMP + 2H2O ΔH4 = -47.1 Cal/mole (4) This large exothermic ΔH clearly makes a significant contribution to driving the over-all reaction toward thymidylate formation. In order to assist in our understanding of the driving forces acting on Reactions 1 to 4, a series of ancillary measurements were made. These are: CH2(OH)SCH2CH2OH + THF ⇄ CH2 - THF + HOCH2CH2SH + H2O ΔH5 = -15.4 Cal/mole (5) and CH2(OH)2 + HOCH2CH2SH ⇄ CH2(OH)SCH2CH2OH ΔH6 = -9.6 Cal/mole (6) These various enthalpy values were combined with free energy values from the literature in a manner which allowed an estimate of the minimum equilibrium constant for Reaction 3 to be obtained. This value, K ≃ 1025, presents a thermodynamic explanation for the failure to demonstrate reversibility for this system. Consideration of the sign of the entropy change accompanying Reaction 3 indicates that the equilibrium constant may be several orders of magnitude greater than this minimum value.