Origin of the slow-binding inhibition of aldolase by D-glycero-tetrulose 1-phosphate (D-erythrulose 1-phosphate) from the comparison with the isosteric phosphonate analog

The mechanistic reaction pathway for the slow-binding inhibition of rabbit muscle aldolase by D-glycero-tetrulose 1-phosphate (D-erythrulose 1-phospha te) was investigated through the use of its phosphonomethyl isoster 4 which was synthezised for this study. The latter is not a substrate nor a slow-b inding inhibitor but interferes in the enzyme-catalyzed reaction with the s ubstrate fructose 1,6-diphosphate in a competitive manner. It was found tha t phosphonate 4 forms an iminium ion with aldolase and undergoes subsequent alpha-proton abstraction to form an intermediate. We show from these resul ts that enzyme slow-binding inhibition by D-erythrulose 1-phosphate is cons istent with a phosphate beta-elimination reaction through the enamine inter mediate. This mechanism takes into account the stereochemical features know n for aldolase, the parallel between enzyme activity recovery and phosphate release after action of D-erythrulose 1-phosphate, and also the same react ion from dihydroxyacetone phosphate.