Skeletal Alkaline Phosphatase

Abstract

The skeletal isoenzyme of human alkaline phospha-tase was partially purified from the serum of a patient with Paget's disease. The pH dependence of the reac-tion with p-nitrophenyl phosphate (PNPP) suggests that either the reaction mechanism involves 2 dissoci-able 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-homoargi-nine, 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 in-creased K,al,P for each of these inhibitors. Caffeine, the-ophylline, and isobutylmethylxanthine were also un-competitive 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 isobutylmethylxan-thine 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 phospha-tase 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. Human serum normally contains a mixture of organ-specific isoenzymes of alkaline phosphatase (EC 3.1.3.1). These are derived from the liver, intestine, bone, and placenta (1, 2). The skeletal isoenzyme of alkaline phosphatase has been associated with the process of bone formation and minerali-zation since 1923 (3, 4), and in patients with Paget's disease,