The pH variation of primary 18O and primary and secondary deuterium isotope effects has been determined by the use of the equilibrium perturbation method for the reaction catalyzed by fumarase. The primary 18O effect is 1.073 from the malate side at pH 5 (with an equilibrium 18O effect of 1.033) but decreases rapidly to near unity above pH 7. The primary deuterium isotope effect is near unity at pH 5 and 9 but is strongly inverse at neutral pH (0.915 from the malate side at pH 7, compared to the equilibrium isotope effect of 0.98). Secondary isotope effects with dideuterated substrates from (Brant malate side were 1.31 at pH 5-6 but decreased to a value of 1.08 at pH 9.6 (the equilibrium isotope effect is 1.45). These data are interpreted to mean that the 3R proton of malate is transferred to a group (probably carboxyl) on the enzyme with a fractionation factor relative to water of at least 1.2, to give a carbanion intermediate with an aci-carboxylate structure which is tetrahedral at C-2 and trigonal at C-3. Carbon-oxygen bond cleavage accompanied by proton transfer from a group (probably imidazole) on the enzyme then gives water and fumarate. By quantitative analysis of the isotope effects, partition ratios for forward and reverse reaction of the E-malate, EH-carbanion, and EH-H2O-fumarate intermediates are calculated as a function of pH. The commitments to catalysis of malate, fumarate, water, and the proton on the enzyme are small at pH 5, and carbon-oxygen bond breaking is totally rate limiting. At neutral and high pH, however, the commitment factors (except that for water) are large, so that no 18O isotope effect is seen, and the other isotope effects are equilibrium ones, with the exact value seen depending on the ratio of forward and reverse commitments.
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