Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance

Biochemistry. 1991 May 7;30(18):4581-5. doi: 10.1021/bi00232a031.

Abstract

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 (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Dihydroxyacetone Phosphate / metabolism
  • Glyceraldehyde 3-Phosphate / metabolism
  • Kinetics
  • Lactoylglutathione Lyase / metabolism
  • Muscles / enzymology*
  • Mutation
  • Pyruvaldehyde / metabolism
  • Rabbits
  • Thiolester Hydrolases / metabolism
  • Triose-Phosphate Isomerase / genetics
  • Triose-Phosphate Isomerase / metabolism*

Substances

  • Glyceraldehyde 3-Phosphate
  • Dihydroxyacetone Phosphate
  • Pyruvaldehyde
  • Thiolester Hydrolases
  • hydroxyacylglutathione hydrolase
  • Lactoylglutathione Lyase
  • Triose-Phosphate Isomerase