Irreversible inhibition of pig kidney copper-containing amine oxidase by sodium and lithium ions

Copper amine oxidase was found to be inhibited in a complex way by small al kali metal ions. Classic enzyme kinetic studies showed that Li+ and Na+ wer e weak noncompetitive inhibitors, whereas the larger alkali metals K+, Rband Cs+ were not inhibitors. However, freezing in the presence of Na+ or Li + surprisingly resulted in complete and irreversible inactivation. In the c ase of Li+, it was possible to show that one ion per subunit was retained p ermanently in the inactivated enzyme, suggesting a structural rearrangement . The mechanism of inhibition was studied using a wide range of spectroscop ic and analytic techniques. Only minor changes in the protein structure cou ld be detected, except for a significant change in the geometry of the copp er site. The unique topaquinone, cofactor was apparently functional and abl e to proceed through the reductive half of the catalytic cycle, but the enz yme no longer reacted with oxygen. The effect of Na+ and Li+ was source-spe cific for pig kidney and bovine kidney amine oxidases, while the enzymes fr om bovine serum or plants were not inactivated, consistent with a mechanism dependent on small structural differences. A model for irreversible inacti vation is proposed in which the cofactor is co-ordinated directly to copper , in analogy with the inactivation reported for Escherichia coli amine oxid ase under crystal growth conditions.