The Pre-steady State of the Myosin-Adenosine Triphosphate System<subtitle>V. Evidence for a Phosphate Exchange Reaction between Adenosine Triphosphate and the “Reactive Myosin-phosphate Complex” <xref ref-type="fn" rid="fn1"><sup>*</sup></xref></subtitle>

Abstract

In our previous work, it was shown that the initial reaction between myosin-ATPase [EC 3.6.1.3, ATP phosphohydrolase] and ATP produces one mole of “reactive myosin-phosphate complex” (EP) per 4×105g of myosin, which is TCA**-labile. However, there has been found so far no unequivocal evidence for a covalent bonding of phosphate to myosin in the complex. In the present work, therefore, a search was made for a phosphate exchange reaction between ATP and EP. The amount of TCA-labile γ32P1 liberated was measured as a function of time after the reaction was started by adding γ-32 P-labelled ATP to myosin in the ratio of 1 mole to 4×105g (the first, or control, experiment). In a subsequent experiment, 1 mole of non-labelled ATP was added to 4×105 g of myosin, and at an appropriate time thereafter, a small amount of γ-32P-ATP, with about the same radioactivity as that of the ATP-32P used in the control experiment, was added to the myosin-ATP system (the second experiment). The 32P1-liberation was followed after adding TCA to stop the reaction, and was compared with that in the control experiment. There are several lines of evidence either that the Michaelis complex (ES) is in equilibrium with myosin (E) and ATP (S), or that the amount of ES is negligibly small. Therefore, the time-course of TCA-labile 32P1-liberation from the myosin-ATP system in the second experiment can be easily calculated from the time-course of the control experiment, if it is assumed that there is no phosphate-exchange reaction between EP and ATP. The difference between the experimental values and the ones calculated on this assumption can be attributed to the exchange of phosphate between EP and γ-32P-ATP. Myosin was preincubated in 2.8 M KC1 and 10 mM MgCl2 at pH 7.5 and 0°C, prior to the addition of ATP, and it was found that the initial reaction of the myosin-ATP system was not affected by preincubation lasting up to, at least, 3 hr. The time-course of TCA-labile 32P1-liberation in the second experiment was insensitive to a variation in the amount of hot ATP (of constant radioactivity) from 0.005 to 0.05 moles per 4×105 g of myosin, when this hot ATP was added to the system one minute after the addition of 1 mole of cold ATP per 4×105 g of myosin. Therefore, the amount contained in EP of an intermediate exchanging P with the terminal P of ATP could be measured by the method mentioned above. In the presence of 2.8 M KC1 and 10 mM MgCl2 and at p11 7.5 and 0°C, 15–20 percent of the total EP were found to be P-exchangeable with ATP 1 min after the addition of ATP to myosin at a molar ratio of 1: 1. The content of phosphate-exchangeable intermediate in the total EP decreased with decreasing concentrations of KCl and MgCl2: it was 0–10 per cent of EP in the presence of 1.08 M KCI and 5 mM MgCl2, and varied considerably from one preparation to another. It was not affected when the temperature was raised from 0 to 15°C, or when 0.1 mM CaCl2 or 0.1 mM EGTA was added to the reaction mixture. In the presence of 1.1M KCI and 5mM MgCl2 and at pH7.5 and 0°C, the time-course of TCA-labile 32P1-liberation was followed, first, with a system in which 1 mole of hot ATP was added per 4×105 g of myosin, and second, with the systems in which 1 mole of hot ATP per 4×105 g of myosin was added various times after the addition of 1 mole of cold ATP. In this experiment a myosin preparation was used which showed no exchange reaction between EP and ATP under the above conditions. In this case, the initial rate of 32P1-liberation was proportional to the amount of total E minus EP, when corrections were made for the effect of ADP and the rate of ATP-decomposition via simple hydrolysis. Therefore, by analyzing the experimental results described above, the life-time of EP could be easily calculated, and it could be shown that the rate constant of decomposition of EP into myosin and P1 was about 1/5 that of simple hydrolysis of ES.