Mechanistic comparison of the cobalt-substituted and wild-type copper amine oxidase from Hansenula polymorpha.
A recent report by Mills and Klinman [Mills, S. A., and Klinman, J. P. (2000) J. Am. Chem. Soc. 122, 9897-9904] described the preparation and initial characterization of a cobalt-substituted form of the copper amine oxidase from Hansenula polymorpha (HPAO). This enzyme was found to be fully catalytically active at saturating substrate concentrations, but with a K(m) for O(2) approximately 70-fold higher than that of the copper-containing, wild-type enzyme. Herein, we report a detailed analysis of the mechanism of catalysis for the wild-type and the cobalt-substituted forms of HPAO. Both forms of enzyme are concluded to utilize the same mechanism for oxygen reduction, involving initial, rate-limiting electron transfer from the reduced cofactor of the enzyme to prebound dioxygen. Superoxide formed in this manner is stabilized by the active site metal, facilitating the transfer of a second electron and two protons to form the product hydrogen peroxide. The elevated K(m) for O(2) at the dioxygen binding site in Co-substituted HPAO, relative to that of wild-type HPAO, is proposed to be due to a change in the net charge at the adjacent metal site from +1 (cupric hydroxide) in wild-type enzyme to +2 (cobaltous H(2)O) in cobalt-substituted HPAO.
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Stephen A
SA
Mills
Yoshio
Y
Goto
Qiaojuan
Q
Su
Julie
J
Plastino
Judith P
JP
Klinman
D006631:Amine Oxidase (Copper-Containing) / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003035:Cobalt / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010843:Pichia / Q000201:enzymology*; D011868:Radioisotopes / Q000378:metabolism; D017186:Titrimetry; D014783:Viscosity
D011868:Radioisotopes; D003035:Cobalt; D003300:Copper; D006631:Amine Oxidase (Copper-Containing); D010100:Oxygen