The effects of buffer cations on interactions between mammalian copper-containing amine oxidases and their substrates.
We and others have observed that substrates for copper-containing amine oxidases cause substrate inhibition at high concentrations. Through use of a novel "pseudoquantitative" rapid equilibrium approach, kinetic analyses with human and bovine enzymes indicate that these effects are consistent with substrates binding to oxidised and reduced enzyme forms. Small cations compete with binding of substrates to oxidised and reduced enzyme, influencing both substrate turnover and substrate inhibition patterns. Cations reduce affinity of the resting bovine enzyme for spermidine, but not benzylamine, indicating that the predominant effect of cations on substrate oxidation results from binding to an anionic site outside the active site. However, binding of cations to the active site of the reduced form of both enzymes attenuates substrate inhibition with both spermidine and benzylamine. Our observations have significant practical implications for researchers assaying kinetic behaviour of these enzymes, and particularly those developing novel inhibitors of human copper-containing amine oxidases.
Holt
A
A
Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada. aholt@pmcol.ualberta.ca
Degenhardt
O S
OS
Berry
P D
PD
Kapty
J S
JS
Mithani
S
S
Smith
D J
DJ
Di Paolo
M L
ML
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001596:Benzylamines / Q000737:chemistry / Q000378:metabolism; D002021:Buffers; D020134:Catalytic Domain / Q000502:physiology*; D002412:Cations / Q000737:chemistry* / Q000494:pharmacology; D002417:Cattle; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D003300:Copper / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D010084:Oxidation-Reduction / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D013095:Spermidine / Q000737:chemistry / Q000378:metabolism
D001596:Benzylamines; D002021:Buffers; D002412:Cations; D015815:Cell Adhesion Molecules; D003300:Copper; C030796:benzylamine; C076668:AOC3 protein, human; D006631:Amine Oxidase (Copper-Containing); D013095:Spermidine