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J. Biochem, 1970, Vol. 67, No. 2 291-295
© 1970 Japanese Biochemical Society

research-article

Effect of pH on the Kinetic Parameters of Yeast L(+)-Lactate Dehydrogenase (Cytochrome b2)

HARUO SUZUKI and YASUYUKI OGURA

Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo Bunkyo-ku, Tokyo

The effect of the pH value on the rate of the overall reaction catalyzed by L(+)- lactate dehydrogenase [EC 1. 1. 2. 3] was studied spectrophotometrically using ferricyanide as electron acceptor. The rates of reduction of the FMN and protoheme of the enzyme with L-lactate at various pH values were measured by the "stopped flow method" using ferricyanide as acceptor. From the relationships between the kinetic parameters and the pH value, the acidic pK-values were found to be 5.3 and 6.0 and the alkaline ones to be 8.8 and 9.7. It was concluded that two kinds of ionizable groups in the enzymeunit are essential for activity, that shifts of the pK-values occur on binding of the substrate to the enzyme-unit, and that in the overall reaction the hydrogen transfer reaction from the substrate to the bound FMN is inhibited by the association and dissociation of protons of the essential groups.


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