Initial and Equilibrium 18O, 14C, 3H, and 2H Exchange Rates as Probes of the Fumarase Reaction Mechanism

• Published in: The Journal of biological chemistry Vol. 244; no. 22; pp. 6270 - 6279
• Main Authors: Hansen, J N, Dinovo, E C, Boyer, P D
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 25-11-1969
• Subjects: Carbon Isotopes; Chemical Phenomena; Chemistry; Deuterium; Fumarates; Hydro-Lyases; Kinetics; Malates; Mathematics; Oxygen Isotopes; Tritium; Water
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)63533-1

Net conversion of malate to fumarate as measured by the A 260 of fumarate is accompanied by a nearly equivalent loss of 3 H or 2 H from (2S, 3R)-3 2 or 3 H-malate to water but by an appreciable incorporation of 18 O from water into unreacted malate. At equilibrium with high substrate concentrations, exchange of the hydroxyl oxygen of
malate with water is faster but interchange of the methylene hydrogen is slower than the carbon skeleton interchange between
malate and fumarate. Relative values for the 18 O (malate) ⇄ O (water) , 14 C (malate) ⇄ C (fumarate) , 3 H (malate) ⇄ H (water) , and the 2 H (malate) ⇄ H (water) exchange rates are 4.0, 2.5, 1.0, and 1.0, respectively. Exchange rate differences and 18 O incorporation into malate as compared to net reaction are accentuated in a glycerol-water medium. The 14 C: 3 H exchange ratio approaches a limiting value of below 1 at low substrate concentrations and a limiting value of between 2
and 3 at high substrate concentrations. Addition of 0.3 m ammonium sulfate accelerates the 3 H exchange, presumably by facilitating proton dissociation from the enzyme. The exchange patterns are consistent with a carbonium
ion mechanism. Theoretical equations and a reaction scheme in accord with the results are given. These allow calculation of
distribution patterns of enzyme-bound intermediates and ratios for a number of velocity constants.