The p-nitrophenyl phosphatase activity of muscle carbonic anhydrase

Abstract

Carbonic anhydrase III from rabbit muscle, a newly discovered major isoenzyme of carbonic anhydrase, has been found to be also a p-nitrophenyl phosphatase, an activity which is not associated with carbonic anhydrases I and II. The p-nitrophenyl phosphatase activity has been shown to chromatograph with the CO 2 hydratase activity; both activities are associated with each of its sulfhydryl oxidation subforms; and both activities follow the same pattern of pH stability. This phosphomonoesterase activity of carbonic anhydrase III has an acidic pH optimum (<5.3); its true substrate appears to be the phosphomonoanion with a K m of 2.8 m m. It is competitively inhibited by the typical acid phosphatase inhibitors phosphate ( K i = 1.22 × 10 −3M), arsenate ( K i = 1.17 × 10 −3M), and molybdate ( K i = 1.34 × 10 −7M), with these inhibitors having no effect on the CO 2 hydratase or the p-nitrophenyl acetate esterase activities of carbonic anhydrase III. The p-nitrophenyl acetate esterase activity of carbonic anhydrase III, on the other hand, has the sigmoidal pH profile with an inflection at neutral pH, typical of carbonic anhydrases for all of their substrates, and is inhibitable by acetazolamide (a highly specific carbonic anhydrase inhibitor) to the same degree as the CO 2 hydratase activity. The acid phosphatase-like activity of carbonic anhydrase III is slightly inhibited by acetazolamide at acidic pH, and inhibited to nearly the same degree at neutral pH. These data are taken to suggest that the phosphatase activity follows a mechanism different from that of the CO 2 hydratase and p-nitrophenyl acetate esterase activities and that there is some overlap of the binding sites.