Kinetic Studies on Gluc-amylase
III. The Influence of pH on the Rates of Hydrolysis of Maltose and Panose
1966 年 59 巻 5 号 p. 469-475
詳細
1966 年 59 巻 5 号 p. 469-475
1. The dependence of the apparent Michaelis constant Km and the apparent maximal velocity V on pH has been studied at 25°C over the pH range from 2.2 to 6.9 for the hydrolytic reaction of maltose and panose catalyzed by crystalline glue-amylase of Rh. delemar.
2. The ionization constants of the essential ionizable groups 1 and 2 of the free enzyme (uncombined with substrate), Ke1, and Ke2, have been determined for maltose and panose as substrate. The values of pKe1, and pKe2 were found to be 2.9 and 5.9, respectively, for the both substrates. The heats of ioniza-tion of the two ionizable groups have been determined for panose to be ΔH1=0kcal/mole and ΔH2=-0.8 kcal/mole. The value of ΔH2 for maltose was equal to that for panose. These results have suggested that the same ionizable groups are involved in the catalysis of the both substrates.
3. The above information, together with that obtained in the previous study on the competition between the two types of sub-strates, has enabled us to conclude that the hydrolyses of the both substrates, and hence both α-1, 4 and α-1, 6 glucosidic linkages, are catalyzed by a single (the same) active center of the enzyme.
4. From the values of pKe and OH, together with the effect of solvent on pKe the essential ionizable groups 1 and 2 of the enzyme have been considered to be both carboxyl groups, the active forms of which are COO- and COOH, respectively. The role of these ionizable groups in the reaction have been discussed.
The authors wish to acknowledge with gratitude Professor J. Fukumoto and Dr. Y. Tsujisaka of Osaka Municipal Technical Research Institute ' for their donation of the crystalline enzyme and Professor K. Shibasaki of Tohoku University for his gift of panose.
