OXONASE AND ESTERASE-ACTIVITIES OF ERYTHROCYTE CARBONIC-ANHYDRASE

The term “oxonase” is derived directly from the common names given to pesticides such as dialkyl p-nitrophenyl phosphates (alkyl paraoxons) and O,O-dialkyl S-( 1,2-dicarbethoxy)ethyl phosphorothiolates (malaoxons) and should be distinguished from “phosphatase” which generally refers to enzyme-catalyzed hydrolysis of phosphate monoesters. 2 Caution: Care should be exercised at all times in the handling of dimethyl 2,4-dinitrophenyl phosphate since it may be expected to exhibit powerful anticholinesterase activity. Indeed, Fukuto & Metcalf (1956) found diethyl 2,4-dinitrophenyl phosphate to be the most potent inhibitor of the Fly-brain cholinesterase among 24 diethyl substituted phenyl phosphates tested, although it actually possessed a relatively low contact toxicity presumably owing to its hydrolytic degradation before reaching the site of action. abstract: The present investigation reveals that bovine carbonic anhydrase, in contrast with the serine esterases, effectively catalyzes the hydrolysis of dimethyl 2,4-dinitrophenyl phosphate. It is thus the first well-characterized enzyme to behave unambiguously as an oxonase. The oxonase and esterase activities of bovine carbonic anhydrase toward dimethyl 2,4-dinitrophenyl phosphate and 2,4-dinitrophenyl acetate, respectively, exhibit a number of clear similarities. Both are characterized below pH 9 by typical approximately sigmoidal pH-rate profiles, defined by pkenz values of 7.37 and 7.53, for cach of which maximal activity requires the basic form of the enzyme. In the pH region 6-8, the Michaelis constants describing the interactions of both substrates with bovine carbonic anhydrase are, to a first approximation, linear functions of the respective turnover numbers. The formal dissociation constants, namely, the values of Km extrapolated to zero turnover, of the enzyme-substrate complexes for dimethyl 2.4-dinitrophenyl phosphate and 2,4-dinitrophenyl acetate are 5.8 X 10-3 and 1.4 X 10-3 M, respectively, while the relative efficiencies of the bovine carbonic anhydrase catalyzed hydropses of these two substrates are directly reflected in the values 6.85 and 118 assumed by the ratios kenz/kOH-at the inflexions in the pH-rate profiles. The apparent dissociation constants in the presence of dimethyl 2,4-dinitrophenyl phosphate and 2,4-dinitrophenyl acetate, respectively, for the complex formed between the enzyme and the specific inhibitor acetazolamide, at pH 8.95, are 3.0 X 10-7 and 3.1 X 10-7 M, thereby implicating to a similar extent the participation of the active-site zinc ion of bovine carbonic anhydrase in both oxonatic and esteratic processes. Unlike the noncompetitive pattern consistently observed for carboxylate esters, however, acetazolamide inhibition of the enzyme catalyzed hydrolysis of dimethyl 2,4-dinitrophenyl phosphate is formally competitive in type. Furthermore, the appearance of the rise in activity commonly observed above pH 9 with bovine carbonic anhydrase rate profiles occurs at a point for 2,4-dinitrophenyl acetate over 1.5 pH units higher than that found in the case of dimethyl 2,4-dinitrophenyl phosphate; such a marked substrate dependence argues strongly in favor of the involvement both of a well-defined substrate binding site and of associated conformational changes in the enzyme. © 1978, American Chemical Society. All rights reserved.