Irreversible reaction of 3-amino-1:2:4-triazole and related inhibitors with the protein of catalase

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Abstract
In addition to 3-amino-l,2,4-triazole, a series of related compounds could cause the irreversible inhibition of purified recrystallized catalase preparations in the presence of H2O2. Of all the compounds tested S-methylisothiosemicar-bazide caused the most rapid inhibition. The minimal structure required for inhibitory activity in this series was >N-NH-C(NH2)R, or an isomer of this structure, in which the primary amino group attached to the C atom had to be unsubstituted and R could be S, O or NH. Substances which can be oxidized by catalase-H2O2 complex I, such as ethanol, formate, nitrite, pyrogallol, etc., prevented the occurrence of these inhibitions but could not reverse them once they had taken place. The rate of inhibition at any particular enzyme inhibitor concentration and temperature did not vary from pH 5.5 to 9.0. The effective inhibitors of this series reacted with catalase H2O2 complex II or catalase-methyl hydroperoxide complex II to liberate free catalase. The second-order reaction constant of this reaction at 37[degree] was 0.26 M-l sec.-l for 3-amino-1,2,4-triazole and 2.4 [image] -1 sec,-1 for S-methylisothiosemi-carbazide. In addition to the irreversible inhibition, 3-amino-l,2,4-triazole reversibly inhibited catalase activity. The directly determined equilibrium constants for the possible reversible reactions of this inhibitor with either free catalase or the primary complex were respectively 40 m[image] and 20 m[image] at 22[degree]. A plot of the logarithm of the catalase activity against time during the irreversible inhibition of catalase by 3-amino-l,2,4-triazole in the presence of an excess of H2O2 gave a sigmoid curve. The possible reasons for such a curve are discussed. Two independent kinetic equations for the irreversible reaction of the inhibitors with catalase-H2O2 complex I were derived. These equations fit the kinetic data obtained on the initial rates of the irreversible inhibition reaction and on the reversible inhibition of catalase activity. For 3-amino-l,2,4-triazole the second-order reaction constant for the irreversible reaction with complex I was about 25 m[image] -l sec.-1, at 22[degree], calculated from either equation. Catalase which had been irreversibly inhibited by 3-amino-l,2,4-triazole had incorporated approximately 1 mole of inhibitor for each mole of catalase hematin which had reacted. On liberation of the hematin by acid-acetone, the incorporated inhibitor remained attached to the protein.