Formation and Decomposition of a Phosphorylated Intermediate in the Reaction of Na+-K+ Dependent ATPase

• Published in: Journal of biochemistry (Tokyo) Vol. 67; no. 5; pp. 693 - 711
• Main Authors: KANAZAWA, TOHRU, SAITO, MASAYUKI, TONOMURA, YUJI
• Format: Journal Article
• Language: English
• Published: Oxford University Press 01.05.1970
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a129297

A simple mixing apparatus driven by solenoids was devised to follow a rapid reaction. The partial reactions of Na+-K+ dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) were separately investigated in the presence of 1mM MgCl2 and monovalent salts at pH8.5 and 15°C, and the following results were obtained. When the phosphorylation reaction was stopped by adding excess EDTA to the reaction medium, the concentration of phosphorylated protein, (EP), decreased exponentially with time. The first-order rate constant of the decrease in EP, kd, increased with increase in KCl concentration (0.50 and 2.26 sec−1, respectively, in the absence and presence of 0.6 mM KCl), and was independent of the concentrations of NaCl and ATP. The rate constant, kd, of the decrease in E32P after the addition of excess unlabelled ATP was essentially the same as that observed after the addition of EDTA. The ratio of the rate of ATP hydrolysis to the EP concentration in the steady state, vo/(EP), increased markedly with increase in KCl concentration (0.57 and 4.43 sec−1, respectively, in the absence and presence of 0.6 mM KCl), but was unaffected by changing the ATP concentration. In general, the ratio, vo/(EP), differed from kd. It was nearly equal to kd in the absence of KCl, but it increased more rapidly than kd with increase in the KCl concentration. In the presence of 0.6 mM KCl, it was twice kd. The amounts of P1 liberated after the interruption of the phosphorylation reaction by the addition of EDTA were about 1.2 and 2.2 times those of the decrease in EP concentration in the absence and presence of 0.6 mM KCl, respectively. The observed time-course of P1-liberation in the initial phase was in good agreement with that calculated from the observed time-course of EP formation, assuming that EP is an intermediate in the reaction and that its specific turnover rate is equal to the observed value of vo/(EP) in the steady state. Guggenheim plots of EP formation in the presence of 140 mM NaCl gave straight lines at a moderate concentration of ATP. The first-order rate constants calculated from the slopes were 2.6 and 4.3 sec−1, respectively, in the absence and presence of 0.6 mu KCl. Even at fairly low concentrations of ATP (0.17 and 0.057μM), EP formation proceeded linearly with time without showing any measurable lag phase (less than 0.05 sec). A Lineweaver-Burk plot of the initial rate of EP formation gave a straight line over a low concentration range of ATP. The maximum rate of EP formation, Vf, and the Michaelis constant, Kf, obtained from the line were 2.36 moles EP/107g sec and 3.6 μM, respectively, in the presence of 140mM NaCl. At higher concentrations of ATP, the double reciprocal plot of the initial rate against (ATP)showed downward deviation from a straight line. The initial rate with 500μM ATP was higher than 15.7 moles EP/107g sec. The rate of EP formation decreased markedly with decrease in NaCl concentration. The maximum rate of EP formation, Vf, in the presence of 0.5 mM NaCl was 0.15 moles EP/107g sec, which was much lower than the value, 2.36 moles/107g sec, in the presence of 140 mM NaCl. The value of Kf remained constant over a wide range of the NaCl concentrations. The enzyme was incubated with AT32P in the absence of NaCl, and then an NaCl-EDTA mixture was added. The amount of EP increased only slightly after addition of the NaCl-EDTA mixture, though it increased significantly in the presence of NaCl without EDTA. A Lineweaver-Burk plot of the steady state rate of ATP hydrolysis in the presence of 140 mM NaCl and 0.6 mM KCl gave two straight lines intersecting at about 5.6μM ATP. The maximum rates, of the overall reaction, Vo, and the apparent Michaelis constants, Ko, were 1.48 moles P1/107g sec and 0.93 μM, respectively, over the low concentration range of ATP, and were 4.08 moles P1/107g sec and 10.7 μM, respectively, over the high concentration range of ATP. The above observations could be reasonably explained by the following reaction mechanism;