FRUCTOSE DIPHOSPHATE ALDOLASE FROM FUSARIUM OXYSPORUM f. LYCOPERSICI: III. STUDIES ON THE MECHANISM OF REACTION

Abstract

Frnctose-1,6-diphosphate aldolase from Fusarium oxysporum f. lycopersici is inhibited by acetylimidazole. The inhibition is reduced in the presence of fructose diphosphate or DL-glyceraldehyde-3-phosphate, but not in the presence of dihydroxyacetone phosphate. Under conditions of almost total inactivation, as measured by the rate of cleavage of fructose diphosphate, the decrease in the rate of exchange is only 18%. These results indicate that the amino acid residues which are altered under these conditions are concerned with the D-glyceraldehyde-3-phosphate rather than the dihydroxyacetone phosphate active site.The rate of cleavage of fructose diphosphate in D₂O decreases, with increasing pH, more rapidly than the rate in H₂O and resembles the decreased rate of tritium exchange into dihydroxyacetone phosphate with increasing pH. These results suggest that a correlation exists between the isolated proton exchange and cleavage reactions and that the decreased rate of exchange and cleavage with increasing pH is due to a decreased rate of proton neutralization of the enzyme – dihydroxyacetone phosphate anion. The addition of acetaldehyde to the aldolase assays at pH 8.0 and pH 9.0 stimulates the cleavage rate of fructose diphosphate to a level normally obtained at pH 7.5, confirming the supposition that the release of dihydroxyacetone phosphate becomes rate limiting with increasing pH. The results obtained by following the cleavage in D₂O also indicate that general base catalysis is involved in the proton neutralization step and that the pK of the general base is less than pH 6.0. The evidence suggests that the general base is either a β-COOH of aspartate or a γ-COOH of glutamate.