Effect of Azide on Some Spectral and Kinetic Properties of Pig-Plasma Benzylamine Oxidase.

The effect of azide on optical absorption and electron paramagnetic resonance (EPR) spectra of pig-plasma benzylamine oxidase has been examined and correlated with the effect of azide on the catalytic activity of the enzyme. A strong reversible interaction between azide and copper in benzylamine oxidase, corresponding to complex-formation with a dissociation constant less than 0.2 mM, was detected by EPR techniques. This interaction is competitive with the EPR-detectable binding of cuprizone to copper in the enzyme, but is not affected by reactions involving the active-site pyridoxal phosphate and has no evident effect on enzyme activity. A weaker reversible interaction between azide and protein-bound copper, corresponding to complex formation with a dissociation constant of 40 mM, was detected spectrophotometrically and by EPR techniques. An absorption band centered at 390 nm is associated with the latter complex, the apparent stability of which was found to be unaffected by a 20-fold variation of the oxygen concentration as well as by reduction of the enzyme with substrate. Azide acts as an uncompetitive inhibitor of benzylamine oxidase with an inhibition constant close to 40 mM. 100 mM azide has essentially no effect on the reactivity of the active-site pyridoxal phosphate towards phenylhydrazine or benzylamine. It is concluded that formation of the enzyme · azide complex exhibiting the 390 nm absorption band precludes reoxidation of the substrate-reduced form of the enzyme, indicating that at least one of the two copper ions in benzylamine oxidase may have a direct catalytic function in relation to the reoxidation process. [ABSTRACT FROM AUTHOR]