Human skeletal alkaline phosphatase. Kinetic studies including pH dependence and inhibition by theophylline.

The skeletal isoenzyme of human alkaline phosphatase was partially purified from the serum of a patient with Paget’s disease. The pH dependence of the reaction with p-nitrophenyl phosphate (PNPP) suggests that either the reaction mechanism involves 2 dissociable residues on the enzyme (8.75 > pK1 > 8.2; 9.35 > pKz > 8.75), or that the monoanion is the active form of the phosphoryl substrate. In this reaction, Tris buffer decreased the apparent value of Km,.NI,,., acting as a nonessential activator. At pH 10, Mg’ increased Vmax without affecting Kml.N,.I.. M&’ binding is determined by a residue on the enzyme with pK = 10.2. Phosphate, phosphoethanolamine, and phenylphosphonate were competitive inhibitors with respect to PNPP, and Tris buffer reduced the value of Ki,,,, relative to carbonate buffer, for each. These inhibitions were pH-dependent. Uncompetitive inhibitions observed with L-homoarginine, histamine, and imidazole were pH-independent (Ki = 2.2 to 2.5 n m , 3.8 to 4.3 mM, and 4.3 to 4.5 n m , respectively, in carbonate buffer), and Tris buffer increased K,al,P for each of these inhibitors. Caffeine, theophylline, and isobutylmethylxanthine were also uncompetitive inhibitors with respect to PNPP; Ki = 18 mM, 0.14 to 0.2 mM, and 0.6 to 0.85 mM, respectively. Inhibitions by theophylline and isobutylmethylxanthine were independent of pH and of Tris (caffeine was not tested). Cyclic CMP was also an uncompetitive inhibitor; Ki = 3.2 to 3.3 m, and independent of pH and of Tris. Cyclic AMP was less effective; Ki = 23 mM. These results indicate that skeletal alkaline phosphatase expresses at least three binding sites during the reaction with PNPP, in addition to the site for M&+. One site for the phosphoryl substrate may require the monoanion, and two others, which are independent of each other and appear only after the phosphate site is occupied, can be occupied by Tris (histamine, etc.) and theophylline (cyclic CMP, etc.), respectively.