The pH-Effect on the Rate of Reaction of Yeast L (+)-Lactate Dehydrogenase

抄録

The pH-effect on the overall reaction rate of yeast L (+)-lactate dehydrogenase [EC 1. 1. 2. 3] was investigated in the pH-range from 5.0 to 9.0, with due precaution to avoid the occurrence of irreversible loss of activity.
When methylene blue, thionine, 2, 6-dichlorophenol-indophenol or cytochrome c was used as acceptor, V^pH_m/V^op_m-pH relations gave sigmoid curves of the first order on both acidic and alkaline sides (V^pH_m: maximum velocity at a given pH; V^op_m: maximum velo-city at the optimum pH). The pK-values were found to be 5.1 and 9.2 at 15°C, and 5.25 and 8.9 at 25°C, independently of the kind of acceptors used. The V_pH/Vop-_pH relations were also obtained as sigmoid curves of the first order, (V_pH: velocity at a given pH; V^op: velocity at the optimum pH; both in the presence of a given, non-saturating, concen-tration of substrate). The V_pH/V_op-pH curve on the acidic side shifted toward alkaline side with the decrease of the concentration of substrate added. The pK-values were 5.6 and 5.9 at 15°C in the presence of 1.05 and 0.105 mM L (+)-lactate, respectively. The pK-values obtained on the alkaline side were 9.2 at 15°C independently of the concentration of substrate added. In the case of ferricyanide used as acceptor, however, the pK-values on the acidic and alkaline sides were 6.0 and 9.2 at 15°C independently of the concentrationn of substrate.
The p(K'_m/K_m)-pH relations which were obtained on the acidic side by applying methylene blue, thionine or 2, 6-dichloro-phenol-indophenol as acceptor, were expressed by a formula, K'_m/K_m=(1+[H⁺]/K₁)/(1+[H⁺]/K'₁), where K₁ and K'₁ were assumed to be 10^-6.0 and 10^-5.1M respectively. From the data obtained at 25°C, the values of K₁ and K'₁ were calculated to be 10^-6.15 and l0^-5.25 M by applying Dixon's graphic method. When ferricyanide was used as acceptor, the p(K'_m/K_m)-values were almcst zero independently of the pH in the reaction media.
A reaction scheme was proposed to account for the various kinetic phenomena observed.
The authors wish to express their gratitudes to Prof. Hiroshi Tamiya for his valuable criticism in this work. We are indebted to Mr. T. Nagashima for his technical assistance. Thanks are also due to the Oriental Yeast Co. for the supply of baker's yeast.