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
