Concentration of activated intermediates of the fructose-1,6-bisphosphate aldolase and triosephosphate isomerase reactions

Biochemistry. 1981 Mar 3;20(5):1223-9. doi: 10.1021/bi00508a026.

Abstract

As discovered by Grazi & Trombetta [Grazi, E., & Trombetta, G. (1978) Biochem. J. 175, 361], fructose-1,6-bisphosphate aldolase of rabbit muscle causes the slow formation of inorganic phosphate (Pi) and methylglyoxal when incubated with dihydroxyacetone phosphate (DHAP). In addition, these authors found an acid-labile intermediate in equilibrium with the aldolase-dihydroxyacetone phosphate complexes representing approximately 60% of the enzyme-bound DHAP species. Experiments are reported here which argue that this acid-labile species is the enzyme-bound enamine phosphate or its equivalent that decomposes by beta elimination in acid. A similar mechanism involving an enediol phosphate is proposed to explain a phosphatase action of triosephosphate isomerase that produces methylglyoxal and Pi at the rate of approximately 0.1 s(-1) at pH 5.5. When DHAP with excess isomerase is quenched in strong acid, the formation of Pi indicates that approximately 5% of bound reactant is in the form of enediol phosphate. The remainder of the substrate is about equally distributed between bound forms of DHAP and D-glyceraldehyde 3-phosphate. This equilibrium differs by 300-fold from the appropriate equilibrium in solution. Yeast aldolase, contrary to expectation, does not catalyze formation of inorganic phosphate and methylglyoxal when incubated with DHAP and gives no evidence fro an enediol phosphate intermediate when quenched in acid.

Publication types

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

MeSH terms

  • Animals
  • Carbohydrate Epimerases / metabolism*
  • Fructose-Bisphosphate Aldolase / metabolism*
  • Hydrogen-Ion Concentration
  • Isotope Labeling
  • Kinetics
  • Muscles / enzymology*
  • Oxygen Isotopes
  • Phosphorus Radioisotopes
  • Rabbits
  • Radioisotope Dilution Technique
  • Triose-Phosphate Isomerase / metabolism*

Substances

  • Oxygen Isotopes
  • Phosphorus Radioisotopes
  • Fructose-Bisphosphate Aldolase
  • Carbohydrate Epimerases
  • Triose-Phosphate Isomerase