Studies on the inhibition of bovine plasma amine oxidase by hydrazines Public Deposited


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  • Several aspects of the potent inhibition of bovine plasma amine oxidase (PAO) by hydrazines were investigated by kinetic and preparative means. The inhibition was classified as pseudo-irreversible by the method of Ackerman and Potter (Proc. Soc. Exptl. Biol. Med. 72, 1 (1949), and was found to exhibit Zone B kinetic behavior (Straus and Goldstein, J. Gen. Physiol. 26, 559 (1943)). The constancy of the mole ratio of inhibitor to enzyme which produced 50% inhibition, (I/E)₅₀, for PAO preparations of different degrees of purity demonstrated the unique specificity of these inhibitors for PAO. Inhibitor potency as a function of structure was found to parallel the reactivity of these hydrazines towards carbonyls in model systems. The kinetically determined amount of (unsubstituted) hydrazine which produced 100% inhibition was found to correspond exactly with the reported pyridoxal phosphate (PLP) content of the enzyme. The isolation of a ¹⁴C-labelled EI complex confirmed this stoichiometry. These results, coupled with the spectral observations of Yamada and Yasunobu (J. Biol. Chem. 238, 2669 (1963)) led to the conclusion that the inhibition most likely proceeded from a nucleophilic attack of the hydrazine molecule on the carbonyl of the enzyme's PLP to form a stable azomethine via a transaldiminization reaction. The kinetic competition observed between hydrazines and substrate indicated that they react with PAO at the same site, PLP, thus confirming the proposal originated by Tabor, Tabor, and Rosenthal (J. Biol. Chem. 208, 645 (1954)) that PLP is involved in the active site. Inhibitor potency was found to decrease with increasing N-methyl substitution in a manner which could not be related exclusively to either steric or inductive effects of the substituents, but rather, depended on the presence or absence of a hydrogen alpha to the attacking nucleophilic -NH₂ on the hydrazine molecule. Thus, binding of hydrazines to the catalytic site of PAO may involve a three-point attachment. Therefore, the active site of PAO can be visualized to contain two subsites: one which binds the α-H of hydrazines or substrates by non-covalent forces, which functions to optimally orient the molecule for the chemical reaction at the enzyme's primary site, PLP. The titration of PAO by hydrazines was found to exhibit a biphasic response. Low inhibitor concentrations enhanced PAO activity, but high concentrations inhibited. This apparent homotropic cooperative effect suggested the presence of an allosteric site for the binding of these inhibitors. PAO was found to exhibit anomalous kinetic order with respect to substrate in the presence of hydrazines; v vs. (S) curves were sigmoidal. Normal Michaelis-Menten kinetics were followed in the absence of these inhibitors, indicating that the binding of a hydrazine molecule by the enzyme potentiated an effect which resulted in the binding of more than substrate molecule. High substrate inhibition of PAO was found to conform to the Haldane mechanism. The dissymmetry of v vs. log (S) plots indicated that at high substrate concentrations PAO binds more than two substrate molecules. Thus, PAO may contain an allosteric site for substrate as well as for hydrazines. A hypothetical model is presented which accounts for these experimental observations in terms of the nature and interaction of PAO's inhibitor and substrate binding sites. PAO was found to undergo a time- and concentration-dependent activation in dilute solution at room temperature, pH 7.0, in the presence or absence of hydrazines which could not be attributed to the presence of an endogenous activator or inhibitor (v vs. (E) plots were linear). Gel-filtration experiments revealed that the activation in the absence of hydrazines was not caused by a shift in the monomer- polymer equilibrium or the dissociation of PAO into subunits. Only one species was eluted from the column (which had a molecular weight corresponding to that of the monomer) whether the enzyme was activated or not. This peak was likewise independent of PAO concentration. These results led to the conclusion that the activation of the enzyme in the absence of inhibitor is most likely due to a conformational change. The activation of the inhibited enzyme was found to be greater than that of the "enzyme alone" control; in other words, the inhibition appeared to be reversed. This reversal of inhibition was found to follow first order (with respect to (EI) kinetics indicating that it was caused by the catalytic decomposition of the hydrazine inhibitors by the enzyme.
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