A Study of the Interaction of Acetoacetic Decarboxylase with Several Inhibitors*

Sodium-2-oxo-propanesulfonate (acetone sulfonate) was found to be a competitive inhibitor of acetoacetate carboxy lyase (acetoacetic decarboxylase, EC 4.1.1.4) the KI of which was numerically equal to the K, for acetoacetate at all temperatures investigated. K,,, like Kr in this case is therefore concluded to represent a dissociation constant. AH for the dissociation of either acetoacetate or acetone sulfonate from the enzyme was 7.3 kcal per mole. From the variation of V,,, with temperature, the apparent enthalpy of activation of the enzymic decarboxylation reaction was found to be 11.4 kcal per mole. Acetylacetone was a potent reversible inhibitor of the enzyme whose association with and dissociation from the sensitive sites were perceptibly slow at room temperature. It exhibited both slope and intercept effects in Lineweaver-Burk (12) analyses, the former effects being greater than the latter. Kr was calculated from the slope effects and KII from the intercept effects. Investigation of the effects of temperature on KI and on KII for acetylacetone revealed that KI increased uniformly and moderately with temperatures in the range 14.8-64”, whereas K’, showed an abrupt increase with temperature above 45”. At all temperatures above 45”, KfI was effectively infinite, and acetylacetone was therefore competitive. In the case of nitrate inhibition K’* was greater than Kr and, from the effects of temperature on K’!, AH was found to be 14.3 kcal per mole, while the corresponding value derived from the variation of K1 with temperature was 18.3 kcal per mole. Mixed inhibition studies with acetylacetone, nitrate, and acetone sulfonate indicated identical sites of action for all three. Borohydride reduction of the enzyme in the presence of acetone sulfonate was performed in a manner which allowed quantitative comparisons to be made. On the basis of its facilitation of borohydride inhibition, a K, for acetone sulfonate was obtained which was in agreement with that derived from the classical kinetic analysis. Acetylacetone did not support borohydride inhibition of the