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J. Biochem, 2003, Vol. 134, No. 5 683-690
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

Effect of Nitration on the Activity of Bovine Erythrocyte Cu,Zn-Superoxide Dismutase (BESOD) and a Kinetic Analysis of Its Dimerization-Dissociation Reaction as Examined by Subunit Exchange between the Native and Nitrated BESODs

Hiroshi Oneda and Kuniyo Inouye*

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502

Bovine erythrocyte Cu,Zn-superoxide dismutase (BESOD) is a dimeric enzyme composed of identical subunits associated through unusually strong non-covalent interactions. The state of the unique tyrosyl residue (Tyr 108) of BESOD was examined, and the kinetics of subunit exchange was studied using Tyr 108 as a probe. UV-absorption difference spectra demonstrate that Tyr 108 is exposed to the solvent, and that the accessibilities to ethanol, ethylene glycol, and polyethylene glycol 600 are 53.5, 52.0, and 44.6%, respectively. Tyr 108 was fully nitrated by tetranitromethane. The pKa values of the phenolic hydroxyl group of native and nitrated Tyr 108 were determined to be 11.3 and 7.9, whereas those of model compounds, L-tyrosine and 3-nitro-L-tyrosine, are 9.8 and 6.8, respectively. When the native and nitrated BESODs of an equal concentration were mixed, the hybrid dimer was formed. From the amount of hybrid dimer formed, the rate constant (k–1) of the subunit dissociation at pH 7.8, 25°C was assessed to be (4.17 ± 0.13) x 10–6 s–1. The activation energy of the subunit dissociation at pH 7.8 was determined to be 117 ± 4 kJ·mol–1. The k–1 value remains constant at BESOD concentrations ranging from 0.62 to 6.8 µM and at pH ranging from 6.0 to 10.0, but increased remarkably with a decrease in the dielectric constant of the reaction mixture. It is suggested that hydrophobic interaction may play a significant role in the subunit interaction.

* To whom correspondence should be addressed. Tel: +81-75-753-6266, Fax: +81-75-753-6265, E-mail: inouye{at}kais.kyoto-u.ac.jp


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