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 alkali metal ions. Classic enzyme kinetic studies showed that Li+ and Na+ were weak noncompetitive inhibitors, whereas the larger alkali metals K+, Rb+ and Cs+ were not inhibitors. However, freezing in the presence of Na+ or Li+ surprisingly resulted in complete and irreversible inactivation. In the case of Li+, it was possible to show that one ion per subunit was retained permanently in the inactivated enzyme, suggesting a structural rearrangement. The mechanism of inhibition was studied using a wide range of spectroscopic and analytic techniques. Only minor changes in the protein structure could be detected, except for a significant change in the geometry of the copper site. The unique topaquinone cofactor was apparently functional and able to proceed through the reductive half of the catalytic cycle, but the enzyme no longer reacted with oxygen. The effect of Na+ and Li+ was source-specific for pig kidney and bovine kidney amine oxidases, while the enzymes from bovine serum or plants were not inactivated, consistent with a mechanism dependent on small structural differences. A model for irreversible inactivation is proposed in which the cofactor is co-ordinated directly to copper, in analogy with the inactivation reported for Escherichia coli amine oxidase under crystal growth conditions.
Padiglia
A
A
Department of Sciences Applied to Biosystems, University of Cagliari, Italy.
Medda
R
R
Lorrai
A
A
Paci
M
M
Pedersen
J Z
JZ
Boffi
A
A
Bellelli
A
A
Agrò
A F
AF
Floris
G
G
D000447:Aldehydes / Q000378:metabolism; D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D002412:Cations; D002942:Circular Dichroism; D007668:Kidney / Q000201:enzymology* / Q000378:metabolism; D008094:Lithium / Q000032:analysis / Q000494:pharmacology*; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D012964:Sodium / Q000032:analysis / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013055:Spectrophotometry, Infrared; D013379:Substrate Specificity; D013552:Swine
D000447:Aldehydes; D002412:Cations; D008094:Lithium; D012964:Sodium; D006631:Amine Oxidase (Copper-Containing)