KINETIC-PARAMETERS FOR THE ELIMINATION-REACTION CATALYZED BY TRIOSEPHOSPHATE ISOMERASE AND AN ESTIMATION OF THE REACTIONS PHYSIOLOGICAL SIGNIFICANCE

Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (k(elim)) were determined for reactions at [Enzyme] >> [Substrate]. The second-order rate constant k(Enz) = 10.1 M-1 s-1 was determined from a plot of k(elim) against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] >> [Enzyme], are k(cat) = 0.011 s-1, K(m) = 0.76 mM, and k(cat)/K(m) = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1 000 000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar. This is attributed to offsetting effects of the deletion, which decrease the steady-state concentration of the enzyme-bound intermediate and increase the rate constant for its breakdown to form elimination products. The kinetic parameters were used to calculate a cellular velocity of 0.4 mM methylglyoxal/day for the formation of methylglyoxal from the elimination reaction catalyzed by triosephosphate isomerase. This calculation suggests that an important function for glyoxalases I and II is to metabolize the methylglyoxal that is produced by triosephosphate isomerase.