RATES AND EQUILIBRIA FOR THE INACTIVATION OF MUSCLE ALDOLASE BY AN ACTIVE-SITE DIRECTED MICHAEL REACTION

Compound 1, 4-(4-nitrophenoxy)-2-keto-n-butylphosphoric acid, was synthesized and shown to undergo elimination of p-nitrophenol in aqueous solution at pH 7.0 to form 2-keto-3-butenylphosphoric acid (2) at a rate 50 times greater than hydration to 2-keto-4-hydroxybutylphosphoric acid (4) occurs. A term in the rate expression for l2-– 2 is ascribed to an intramolecular proton abstraction of the a proton by the phosphate oxygen. Rabbit muscle aldolase does not catalyze the production of even 1 equiv of p-nitrophenol in the presence of excess 1. The elimination product, 2, however, causes time-dependent inhibition of the enzyme. The pH and concentration dependence of this reaction was studied, as well as protection from inhibition by 2 using dihydroxyacetone phosphate. Formation of the mixed disulfide of β-mercaptopropionate with an active-site thiol group also affords protection from inhibition by 2. The rate of hydrolysis of the enzyme-inhibitor complex was measured by two methods, and this demonstrated that the equilibrium constant for binding of 2 was IO9M-1at pH 7. Compounds 1 and 4 were shown to bind to aldolase but not undergo reaction. A mechanism for inactivation is proposed in which 2 forms an iminium ion with the lysine function, followed by a Michael-type addition of a thiol to afford the inactive complex. © 1979, American Chemical Society. All rights reserved.