Kinetic studies with muscle acylphosphatase

Abstract

1. 1.|We have established that catalysis of acyl phosphate hydrolyses by muscle acylphosphatase (acyl phosphate phosphohydrolase, EC 3.6.1.7) is competitively inhibited by the orthophosphate product. There is negligible inhibition by the carboxylic acid product, but added buffer anions like chloride or perchlorate do inhibit. 2. 2.|Using acyl phosphatase extracted from chicken muscle, and benzoyl phosphate as substrate, a kinetic analysis employing the integrated rate equation and the pH range 3–12, has provided the values and pH dependencies of the Michaelis parameters at 25°. 3. 3.|The effect of pH on K s (enzyme-substrate dissociation) reveals that PhCO · OPO 3H − is less strongly bound than is PhCO · OPO 3 2−, and that a group in the enzyme, p K a = 7.9, when deprotonated, slightly weakens PhCO · OPO 3 2− binding. p K a for the dissociation of PhCO · OPO 3H − is 4.8 at 25°. 4. 4.|The effect of pH on K p (enzyme-phosphate dissociation) shows that H 2PO 4 − is also less strongly bound than is HPO 4 2−. Both these species, however, are more strongly bound to the enzyme than their benzoylated counterparts. The HPO 4 2− binding is also affected by the group of p K a = 7.9. All the K p and K s values lie within a factor of about 10, centreing on 10 −3 M. 5. 5.|The effect of pH on k (the rate constant of the surface reaction) shows that PhCO · OPO 3H −, although adsorbed, is not readily hydrolysed by the enzyme, and that a group in the enzyme, p K a = 11.0, must be protonated for efficient enzymatic hydrolysis of PhCO · OPO 3 2−. 6. 6.|A complete reaction scheme involving the various substrate, product, and enzyme acid-base ionisations and complexes, leads to a rate equation which correctly reproduces the observed, overall pH dependencies of K s , K p , and k, thus confirming the essential correctness of our analysis. 7. 7.|Our results, together with those of previous studies, lead to a chemical mechanism for the enzymatic catalysis which differs significantly from those of nonenzymatic hydrolyses of acyl phosphates. The substrate is held primarily by the phosphate group. During the surface reaction the phosphorus atom is uniquely located, by bonds to three of the phosphate oxygen atoms, and suffers a slow, nucleophilic attack by an adjacent water molecule. The mechanism explains the observed unsuitability of species ROPO 3 2−, RCO · OPO 3H −, and RCO · OPO 3R′ − as substrates for muscle acylphosphatase.