Proton relaxation study of the hog kidney diamine oxidase active center

Abstract

Proton relaxation studies of the interactions with hog kidney diamine oxidase [EC-1.4.3.6] of H2O, substrate-analog inhibitors, and product analogs indicate that the active site Cu(II) is not located near the oxidizing site of the enzyme, rather near the nonoxidized end of the binding substrate. The studies with histamine derivatives provide evidence for a concentration-dependent occupation of 2 sites. The site which is populated at high concentrations provides proximity of the imadazole ring nitrogen N1 to the Cu(II). H2O binds at the Cu(II) of the native enzyme. This H2O is probably not involved in the hydrolysis of the enzyme-substrate imine bond to eliminate the 1st reaction product. O2 does not compete with H2O for a site on the Cu(II) ion. In the case of one of the probes, namely the NH4 (product) analog dimethylamine, the validity of the protein relaxation results was verified by also observing the nitrogen (15N) relaxation rates of NH4 itself. The conclusion that the ammonium ion is not directly bonded to the active site Cu(II) is reached from both the proton and N relaxation experiments.