Mitochondrial Monoamine Oxidase II. ACTION OF VARIOUS INHIBITORS FOR THE BOVINE KIDNEY ENZYME. CATALYTIC MECHANISM

Abstract Inhibitions of the relatively highly purified flavoenzyme, bovine kidney mitochondrial monoamine oxidase, previously described in Paper I (Erwin, V. G., and Hellerman, L., J. Biol. Chem., 242, 4230 (1967)), by several agents of current interest have been investigated. The inhibitors include N-methyl-N-(2-propynyl)benzylamine (pargyline), (+)-trans-phenylcyclopropylamine (d form of tranylcypromine), as well as various substituted hydrazines including 1-hydrazino-phthalazine (hydralazine) and β-phenylethylhydrazine (phenelzine). With the exception of hydralazine which produced a reversible, substrate-competitive inhibition (Ki, 2 x 10-5 m), the inhibitory hydrazine derivatives investigated caused irreversible inhibitions, differing primarily in the rate of attack on the enzyme; aromatic hydrazines, e.g. phenylhydrazine, were more aggressive than aliphatic hydrazines, e.g. methylhydrazine, members of either class being more active than hydrazides. When graded amounts of arylhydrazine, tranylcypromine, or pargyline were allowed to react with the enzyme, the activity of the enzyme was observed to decrease as a linear function of the amount of inhibitor added; complete inhibition was attained at a ratio of 1 mole of inhibitor per 105 g of protein. It is proposed that these results represent titration at the catalytic site of monoamine oxidase. (+)-Tranylcypromine is known to act as a substrate-competitive inhibitor of monoamine oxidase; for our preparation, Ki was determined as 5.8 x 10-8 m and inhibition was seen to be only partially reversed by dialysis in the presence of substrate. Inhibition by pargyline was not reversible and studies performed with 14C-pargyline (labeled at C-7) indicated that reaction of this inhibitor with monoamine oxidase included ultimate formation of a bond to the flavoenzyme that appeared to be essentially covalent in character. Evidence is presented that suggests that pargyline interacts initially with monoamine oxidase as an electron donor for this flavoenzyme; in a nitrogen atmosphere, reduction of the flavoenzyme by pargyline is followed by or accompanies the irreversible binding step. It was observed that pargyline did not inhibit after the enzyme was first reduced anaerobically with excess benzylamine or with sodium dithionite (followed by addition of benzylamine before the introduction of air). Tranylcypromine, a reversible, nonreducing inhibitor, was found capable of inhibiting the enzyme under such conditions. Analytical determination establishing persistence of approximately 8 sulfhydryl residue equivalents per 105 g of protein in the enzyme initially inhibited by either pargyline or phenylhydrazine (identical with the sulfhydryl content of the native enzyme in our uninhibited preparation) established that these compounds had not effected inhibition of monoamine oxidase through a process involving irreversible interaction with these protein sulfhydryl residues. A mechanism for inhibition of monoamine oxidase by pargyline is proposed. Evidence is cited suggesting that the catalytic mechanism for mitochondrial monoamine oxidase follows a pattern closely related to the mechanism (Neims, A. H., De Luca, D. C., and Hellerman, L., Biochemistry, 5, 203 (1966); Hellerman, L., and Coffey, D. S., J. Biol. Chem., 242, 582 (1967)) recently proposed for the enzyme, d-amino acid oxidase.