Reaction of aortic lysyl oxidase with beta-aminopropionitrile

beta-Aminopropionitrile (BAPN) is a potent irreversible inhibitor of lysyl oxidase, the enzyme which initiates cross-linkage formation in elastin and collagen. The initial interaction of BAPN with aortic lysyl oxidase is competitive with elastin or alkyl amine substrates. Irreversible inhibition develops in a time- and temperature-dependent fashion upon incubation of enzyme with BAPN in the absence of substrate with a limiting inactivation rate constant of 0.16 min-1 and a KI of 6 microM at 37 degrees C. The labeled carbons of [1,2-14C]BAPN and [3-14C]BAPN covalently bind to the enzyme to equivalent extents and in parallel with the development of inactivation, negating the possibility that the nitrile moiety is eliminated from BAPN by enzymatic action. The copper content of the enzyme is not significantly altered upon interaction with BAPN. The extent of labeling by [14C]BAPN is reduced by prior treatment of the enzyme with carbonyl-modifying reagents, suggesting the possibility of enzyme-inhibitor Schiff base formation. However, BAPN is not processed to a free aldehyde product upon incubation with lysyl oxidase. A mechanism of inhibition is postulated which involves the formation of a covalent bond between an enzyme nucleophile and a ketenimine formed from BAPN by enzyme-assisted beta-proton abstraction.