The mechanism of the skeletal muscle myosin ATPase. II. Relationship between the fluorescence enhancement induced by ATP and the initial Pi burst.

A major question about the mechanism of the myosin ATPase is how much of the fluorescence change which accompanies the binding of ATP to myosin is due to the conformational change induced by ATP and how much is due to the subsequent hydrolysis of ATP in the initial Pi burst. Several laboratories have suggested that the maximal rate of the fluorescence change represents the rate of the irreversible conformational change induced by ATP. In the present study, the rate of irreversible ATP binding, the rate of the initial Pi burst, and the rate of the fluorescence enhancement were compared under varied conditions. The results show that: 1) the fluorescence enhancement is mainly due to the hydrolysis of ATP in the initial Pi burst rather than to the conformational change induced by the binding of ATP; 2) the rate of the initial Pi burst is considerably slower than the rate of irreversible ATP binding at high ATP concentration; 3) the rate of the initial Pi burst is almost the same as the rate of the fluorescence enhancement. Therefore, the maximum rate of the fluorescence enhancement represents the rate of the initial Pi burst rather than the rate of the conformational change induced by ATP binding.

A major question about the mechanism of the myosin ATPase is how much of the fluorescence change which accompanies the binding of ATP to myosin is due to the conformational change induced by ATP and how much is due to the subsequent hydrolysis of ATP in the initial Pi burst. Several laboratories have suggested that the maximal rate of the fluorescence change represents the rate of the irreversible conformational change induced by ATP. In the present study, the rate of irreversible ATP binding, the rate of the initial Pi burst, and the rate of the fluorescence enhancement were compared under varied conditions. The results show that: 1) the fluorescence enhancement is mainly due to the hydrolysis of ATP in the initial Pi burst rather than to the conformational change induced by the binding of ATP; 2) the rate of the initial Pi burst is considerably slower than the rate of irreversible ATP binding at high ATP concentration; 3) the rate of the initial Pi burst is almost the same as the rate of the fluorescence enhancement. Therefore, the maximum rate of the fluorescence enhancement represents the rate of the initial Pi burst rather than the rate of the conformational change induced by ATP binding.
In the accompanying paper (3), we presented evidence, in agreement with the work of Taylor (4,5), Trentham (6), Weeds (7) and their collaborators, that both heads of the myosin molecule follow the same biochemical mechanism in their interaction with ATP. Both heads bind ATP irreversibly and then rapidly hydrolyze it in the initial Pi burst. However, certain important details of this mechanism remain unclear. Using the formalism of Bagshaw et al. (8), where M is myosin, T is ATP, D is ADP, Pi is inorganic phosphate, and * and ** qualitatively represent the amount of fluorescence enhancement shown by the various intermediates, the basic mechanism can be written as follows: M+ TzzM.T&M*.T$M**.D.P, (1) F?M*.D.P,~x!M+D+P, One of the key features of this model is that the binding of ATP is postulated to occur in two steps: weak binding of ATP * Preliminary reports of this work have been previously presented (1,2). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
+ To whom reprint requests should be addressed at: Laboratory of Neurochemistry, Bldg. 36, Room 4D-28, National Institute of Neurological Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Md. 20205.
in the collision intermediate (44. Z'), followed by an irreversible conformational change to form M* . T. The major evidence for such a two-step binding process is that at 0.1 M ionic strength, the rate of the fluorescence enhancement which accompanies ATP binding, levels off at high ATP concentration (8). If the binding of ATP were a single step process, the rate of the fluorescence enhancement would be directly proportional to the ATP concentration used. Since the rate of the fluorescence enhancement as well as the rate of H' release level off at high ATP concentration, both Bagshaw et al. (8) and Koretz and Taylor (9) have suggested that these phenomena are caused by the conformational change, M. T + M* . T. However, it is not clear whether all of the fluorescence enhancement is associated with the binding of ATP or whether the subsequent hydrolysis of ATP in the initial Pi burst affects both the magnitude and the rate of the fluorescence change. Werber et al. (10) originally suggested that the fluorescence enhancement was mainly due to the presence of the intermediate, M** . D. P,. Bagshaw et al. (8) and Bagshaw and Trentham (11) suggested on the basis of indirect evidence from analogue studies that approximately half of the fluorescence enhancement observed with ATP was due to M*-T and half to M** . Da Pi. However, they assumed that the transition from M* . T to M* *. De Pi i.e. the initial Pi burst, was faster than the conformational change from M. T to M* . T so that only the rate of the latter conformational change was detected in studies on the fluorescence enhancement.
In contrast, Taylor (5) and Sleep and Taylor (12) reported that the rate of the initial Pi burst was slower than the rate of the ATP binding, i.e. the rate of the transition from M. T to Mt. T. However, because the rate of the fluorescence enhancement was equal to the rate of ATP binding and showed no evidence of being biphasic, Taylor (5) and Sleep and Taylor (12) suggested that all of the fluorescence enhancement was due to the transition from Ma T to M* . T with no fluorescence change accompanying the subsequent initial Pi burst. More recently, however, Johnson et al. (13) also suggested that part of the fluorescence change is due to the initial Pi burst.
In the present study, we investigated the rate of the fluorescence enhancement, the rate of irreversible ATP binding, and the rate of the initial P, burst to determine if the model of Bagshaw et al. (8) for the myosin ATPase is correct. The results show that, in fact, the fluorescence enhancement is due mainly to the hydrolysis of ATP in the initial Pi burst rather than to the binding of ATP. Furthermore, because the rate of the hydrolysis step is slower than the rate of ATP binding at high ATP concentration, the data strongly suggest that the maximum rate of the fluorescence enhancement represents the rate of the initial Pi burst rather than the rate of the conformational change induced by ATP binding.
. Fig. 1A shows, for S-l,' the pseudo-first order rate constant of the fluorescence enhancement as a function of free ATP concentration at 20°C 0.1 M KCl. As can be seen, the rate constant levels off at high ATP concentration which requires that the binding of ATP be at least a two-step process. These data are in agreement with the results first reported by Bagshaw et al. (8). From these results and their ATP analogue binding studies, they concluded that ATP binding occurred in two steps, i.e. (first part of Scheme 1):

RESULTS
k-1 k-2 The fluorescence change was then assumed to be due to the formation of both M* . T and M** e D-P, (see Scheme 1) but since the rate of the formation of M**. D-P,, h3 + h+, in Scheme 1, was assumed to be much faster than h2, the rate of the fluorescence change here depended only on k, and kz. As shown by Bagshaw et al. (8) predicts that a double reciprocal plot of l/hub, uersus l/[rJ will yield a straight line with l/k2 as the ordinate intercept and -k,/k.., as the abscissa intercept (Fig. 1D). However, as discussed by Bagshaw et al. (8), the data in Fig. 1 can also be interpreted by a different two-step mechanism which involves a myosin isomerization step prior to ATP binding, i.e.
To differentiate these two mechanisms, the rate of the fluorescence enhancement was determined as a function of HMM concentration under conditions where the added HMM concentration was much greater than the added ATP concentration. If the mechanism of ATP binding follows Scheme 2, then a double reciprocal plot of l/hot,, versus l/[M] will be identical to the double reciprocal plot of l/k,,,,, versus l/ [ATP] obtained where the added ATP concentration is much greater than the added HMM concentration. This is because in Scheme 2 the HMM and ATP are symmetric, i.e. changes in either species cause the same effect on the rate of the reaction. On the other hand, the integrated equation describing the time course for the consumption of ATP with [HMM] >> [ATP], contains an exponential term and a linear term. Since the observed time course followed a single exponential function, the linear term can be assumed to be constant, so that kohs can be expressed as: Equation 6 indicates that hOhE is a linear function of [Ml. Therefore, a plot of l/k,,,, versus l/ [M] will yield a straight line with an intercept at the origin, quite different than the plot of l/kohs uersus l/ [ATP] shown in Fig. 1B. Fig (solid circles). Similar results were obtained using S-l ( Fig. 2B). In all cases, the straight lines do not intersect the origin. Instead, as predicted by Scheme 2, the double reciprocal plots with either protein or ATP in excess are identical. These data exclude the mechanism (Scheme 4) involving a protein isomerization step prior to ATP binding. Hence, the fluorescence enhancement gssociated with ATP binding is due to a process which follows the binding of ATP.
We next investigated how much of the fluorescence enhancement is due to the conformational change which immediately follows ATP binding (M.T + M*. T), and how much to the subsequent hydrolytic step (M*-T --+ M* * . D. P,). As was pointed out above, in developing Scheme 1 for the mechanism of ATP binding, Bagshaw et al. (8) assumed that the rate constant for the initial P, burst was faster than the rate constant for the conformational change Me T -+ M* . T. On this basis, they suggested that the rate of the fluorescence increase was unaffected by the rate of the initial Pi burst. However, when we performed an experiment similar to the experiment shown in Fig. 1, but at very low ionic strength, the results were inconsistent with those expected for Scheme 2. As shown in Fig. 3A, the value of kohs for the fluorescence enhancement does plateau at high ATP concentration as expected. However, the kohs does not follow a hyperbolic function with respect to ATP concentration as is demonstrated by the nonlinear double reciprocal plot of these data shown in Fig. 3B. This experiment was repeated numerous times and at very low ionic strength the plot was always concave rather than linear. Since kobs levels off at high ATP concentration the data in Fig. 3 suggest that a two-step process occurs when ATP binds to myosin. However, since the double reciprocal plot of hubs uersus ATP is not linear, the data in Fig. 3 also suggest that the first step in the binding of ATP is and the Initial Pi Burst of Myosin  Bagshaw et al. (15), the rate of the initial Pi burst is much faster than hS, Scheme 2 predicts that the rate of irreversible ATP binding and the rate of the fluorescence enhancement will be identical. Scheme 7, on the other hand, predicts that the rate of irreversible ATP binding will be faster than the rate of the fluorescence enhancement which should be essentially equal to the rate of the initial Pi burst. Fig. 4 shows the rate of irreversible ATP binding, the rate of the fluorescence enhancement and the rate of the initial Pi burst at 80 PM ATP where, as shown in Fig. 3, the rate of the fluorescence enhancement is maximal. As can be seen, the rate of irreversible ATP binding is considerably faster than the rate of the fluorescence enhancement or the rate of the initial Pi burst. On the other hand, the rates of the fluorescence enhancement and the initial Pi burst are nearly identical with the rate of the initial Pi burst being only about 20% lower than the rate of the fluorescence enhancement.
The irreversible ATP binding process is 82% complete within 13 ms (Fig. 4A) which corresponds to a kohs > 100 s-l. This is significantly faster than the kobs o f 20 and 23 s-' obtained for the Pi burst and fluorescence enhancement, respectively. This experiment was repeated numerous times under this condition and the rate of irreversible ATP binding was always found to be considerably faster than the rate of the initial Pi burst and the fluorescence enhancement.
It should be pointed out that, as shown in Fig. 4B, the rates of both the irreversible ATP binding and the initial Pi burst tend to decrease somewhat during the last 20% of the reaction. This decrease in rate was usually observed, although a similar decrease in the rate of the fluorescence enhancement was not observed. It is possible that the decrease in the rates of the irreversible ATP binding and the initial P, burst are due to partial denaturation of a small amount of S-l. However, this small deviation does not invalidate the basic finding that the rate of the irreversible ATP binding is considerably faster than the rate of the fluorescence enhancement which, in turn, is nearly equal to the rate of the initial Pi burst as predicted by Scheme 7.
If of M* . T and M* *. D. Pi is detected, and at high ATP concentration where the formation of M* . T becomes so fast that it is not detected due to the dead time of the stopped flow apparatus so that only the formation of M* * . D a P, is observed. Fig. 5 shows that, as predicted by Scheme 7, the magnitude of the fluorescence enhancement is only about 25% lower at high [ATP] than at low [ATP], suggesting that about 75% of the fluorescence enhancement is indeed due to the formation of M**.D-P If Scheme 7 is correct, then at an ATP concentration where the rate of the fluorescence enhancement is about half its maximal value, a lag should be observed in both the rates of the fluorescence enhancement and the initial P, burst, whereas the rate of the irreversible ATP binding should not show a lag. In fact, Bagshaw et al. (15) previously reported that a lag occurred in the initial P, burst. The lag in the rates of the initial P, burst and the fluorescence enhancement is expected because at relatively low ATP concentration, the hydrolysis step (M*-T e M** * D*Pi) with a rate constant, ks + k-3 cannot occur until after the relatively slow second order binding of ATP takes place (M + T F? M*. T) with an observed rate constant of Klkz[ATP]. Fig. 6 shows that a lag in the fluorescence enhancement and the initial P, burst does indeed occur. The lag in the rate of the fluorescence enhancement is slightly smaller than the lag in the initial Pi burst. This is probably due to the small fluorescence increase which occurs on formation of M* . T and the data treatment does not take this into account. Nevertheless, the observation of a lag in the fluorescence enhancement is strong evidence that the Fluorescence Enhancement and the Initial Pi Burst of Myosin increase in fluorescence is associated mainly with the hydrolysis of ATP rather than the irreversible binding of ATP. The comparisons of the rate of irreversible ATP binding, at an ATP concentration where K&[ATP] > kB + Km3 will the rate of the fluorescence change plateau at its maximal value, k3 + LX. But, as the KC1 concentration is increased to 0.1 M at pH = 7, 15'C, Ki122 (the second order binding constant) shows about a 3-fold decrease, while KS + k-s (the rate of the initial P, burst) shows about a 2-fold increase (Table I). Therefore, the ATP concentration must be in the mihimolar range before the maximal fluorescence rate is achieved. Since the initial P, burst cannot be accurately measured at ATP concentrations in great excess of the S-l concentration due to a high blank at this high [Y-~'P]ATP concentration, a comparison of the maximal rate of the fluorescence increase and the rate of the initial P, burst cannot easily be performed at pH 7,0.1 M KCl. However, as shown in Fig. 7, at pH 6.4, 0.1 M KCl, k3 + k-3 is lower than at pH 7 so that the maximal rate of the fluorescence enhancement occurs at a relatively low ATP concentration. Therefore at pH 6.4, we can compare the rate of the initial P, burst, the rate of irreversible ATP binding, and the rate of the fluorescence the rate of the initial Pi burst and the rate of the fluorescence enhancement shown in Figs. 4 and 6 were performed at very low ionic strength. It is of interest to determine whether at higher ionic strength as well, the fluorescence enhancement is associated with the initial Pi burst rather than the binding of ATP. However, there is a difficulty in making this determination at higher ionic strength.  All the experiments were done using S-l. In cases where HMM or myosin were also used, the rates were not found to be significantly different from those obtained using S-l. The value for Klk2 is extracted from the slope of a plot of k "I,, versus ATP, e.g. Fig. lA, where the concentration of ATP is low and the huh, is proportional to the ATP concentration added. The value of k.l + k-1 is obtained from the plateau region of the same plot where the kuh, is no longer dependent on the concentration of ATP added. The values for k% + k-1 at 0.5 M KCl, 20 and 15°C pH 8, could not be accurately determined since the kObh did not reach a maximum value even at 2 mM ATP and at this high concentration of ATP, the magnitude of the fluorescence enhancement becomes very small partly because of the absorption of the excitation light by the ATP and partly because a portion of the maenitude is lost due to the dead time of the stormed flow annaratus.  Fig. 7. The concentrations of S-l and ATP used were 20 and 80 pM, respectively.
The normalized time courses of A are plotted as fiit order plots in B. 0, irreversible ATP binding; A, initial P, burst, 0, fluorescence enhancement. The rates observed were greater than 100 ss' for the irreversible ATP binding, 22 s-' for the initial P, burst, and 26 s-' for the fluorescence enhancement. enhancement at relatively high ionic strength. Fig. 8 shows this comparison at the ATP concentration marked by the arrow in Fig. 7. As can be seen, the same result was obtained under this condition as was found at lower ionic strength at pH 7. The irreversible binding of ATP is considerably faster than the rate of the fluorescence enhancement which in turn is nearly identical to the rate of the initial Pi burst. Therefore, even at relatively high ionic strength, the fluorescence increase appears to accompany the hydrolysis of ATP in the initial P, burst rather than the conformational change induced by the binding of ATP.

DISCUSSION
In this paper we have investigated the cause of the fluorescence increase which accompanies the binding of ATP to myosin. Our initial experiments ruled out the mechanism involving an isomerization of the myosin prior to ATP binding since such a mechanism required the hohs to be directly proportional to the concentration of myosin when the experiments are carried out with the condition that [myosin] > [ATP]. In fact, the rate of the fluorescence increase plateaus at high protein concentration just as the rate of the fluorescence increase plateaus at high ATP concentration. Therefore the process which is responsible for the fluorescence enhancement must occur subsequent to the binding of ATP.
The fundamental pre-steady state kinetic studies of Trentham and his collaborators (6,8,11,15,16) have demonstrated; first, that the binding of ATP is essentially irreversible; second, that a rapid equilibrium occurs between M*. T and M* * . D. P,, which is responsible for the '"0 exchange observed with myosin; and third, that the rate-limiting step of the myosin ATPase is a conformational change occurring after the initial P, burst but before product release. They also found that both with ATP and several ATP analogues the rate of the fluorescence change levels off at high nucleotide concentrations and on this basis they suggested the following mechanism for the fluorescence increase: -50% fluorescence t ks t + M**.D.P, k-3 where Kn > Kz, kmp -0, and k-1 >> hS. In this model, although a fluorescence increase accompanies the hydrolysis of ATP, the rate of the fluorescence change depends only on K, and k2 since hi3 > kz. Taylor (5) and Sleep and Taylor (12)  They made this modification because they found kir + he:< to be less than k2 but the rate of the fluorescence enhancement still appeared to follow a single exponential function. Both of these models predict that 1) the rate of the fluorescence enhancement will proceed via a single exponential, 2) the observed rate constant will follow a hyperbolic function with respect to ATP concentration, 3) the rate of irreversible ATP binding will be equal to the rate of the fluorescence enhancement, and 4) the initial P, burst will occur with at least a slight lag after the fluorescence increase. In support of these models, Taylor (5) and Sleep and Taylor (12) presented data showing that the rate of irreversible ATP binding was equal to the rate of the fluorescence enhancement, which fitted a simple exponential. In addition, they found that the rate of the initial P, burst was about half the rate of the fluorescence enhancement.
The data presented in this paper are in disagreement with the data of Taylor (5) and Sleep and Taylor (12), and suggest that the models for the fluorescence enhancement proposed by Bagshaw and Trentham (11) and Taylor (5) require modification. First, at pH 7 in the absence of KC1 (Fig. 3), we find that the observed rate constant for the fluorescence enhancement is not hyperbolically related to the ATP concentration as these models predict. Second, and most important, our results clearly show that the rate of irreversible ATP binding is considerably faster than both the rate of the fluorescence enhancement and the rate of the initial P, burst. Finally, we find that the rate of the fluorescence enhancement is similar to the rate of the initial P, burst. Frequently, we find that the initial Pi burst tends to be about 15% slower than the rate of the fluorescence enhancement.
However, because these two measurements are carried out on different instruments and are both very temperature-dependent, it is likely that this small difference is within the range of experimental error. Recently, in agreement with the results presented in this paper, Johnson et al. (13) have also reported that a significant amount of the fluorescence increase is associated with the initial P, burst and that the maximal rate of the fluorescence enhancement is equal to the rate of the initial P, burst. On the basis of the data presented in this paper, we conclude that most of the fluorescence enhancement induced by ATP is not due to the conformational change which occurs when ATP binds but rather to the subsequent hydrolysis of the ATP in the initial P, burst. Thus the model of Bagshaw et al. where k-.' << k.], k, >> k.3 and M*. T shows only a small fluorescence increase compared to the formation of M* *. Da P,. In this model the rate of irreversible ATP binding is Klkr while the maximal rate of both the fluorescence increase and the initial Pi burst is k:3 + k-:3. As in the model of Bagshaw et al. (8), in this model two steps are required for the development of fluorescence change, the second order binding of ATP (Klk2) followed by a conformational change due to the hydrolysis of ATP, (k:, + k-:1). But, in this model, the second order binding step is irreversible. Therefore, this model is consistent with the data which show that at low ATP concentration the rate of the fluorescence increase is not a single exponential process (Fig. 6) and that the observed rate constant for the fluorescence enhancement does not show a simple hyperbolic dependence on ATP concentration (Fig.   3B).
In presenting this model, we have assumed that the second order binding of ATP to myosin is, itself, a two-step process involving, first, the equilibrium formation of the collision intermediate M. T and then an irreversible conformational change to form M* . T. Thus, the second order rate constant for ATP binding is K,k? rather than just Iz,. Of course, from our own data we have no direct evidence for a two-step binding process for ATP. Because the irreversible binding of ATP must be measured in the quenched flow apparatus, the shortest reaction time which could be obtained was about 12 ms. At this time more than 80% of the myosin forms a complex with ATP, suggesting that, at 80 pM ATP, the pseudo-first order rate constant for M* . T formation (K&z [ATP]) is about 100 s-', i.e. K&2 -1 x 10" M-l s-l. This is somewhat slower than expected on the basis of the second order rate constant for ATP binding (K&z) obtained from the rate of the fluorescence enhancement measured at very low ATP concentration. This measurement gave a value for Klkz of 3 x 10" M-' sm'.
Thus, it is possible that the pseudo-first order rate constant for M*. T formation is beginning to level off at high ATP concentration.
However, within the limits of accuracy of the measurement we cannot be certain of this. Therefore, we cannot directly determine whether the binding of ATP is a two-step process nor can we determine the value of hp. However, a two-step binding process is certainly not unlikely since a value for Klkz of about 1 X 10" is at least 1 order of magnitude lower than the value expected for a diffusion controlled reaction (16,17). Furthermore, Bagshaw et al. (8) have shown that both ATPyS and ADP bind to myosin in two steps and therefore it seems very likely that ATP also binds in this way.
In this regard, it is useful to consider how our results with ATP relate to the studies of Bagshaw et al. (8) using ADP and ATPyS. At 20°C 0.1 M KCl, pH = 7, they measured, as a function of nucleotide concentration, the rates of the fluorescence change induced by ATPyS, which is not hydrolyzed in an initial Pi burst, and ADP, which, of course, is not hydrolyzed at all. They found that, for both nucleotides, the rates of the fluorescence change showed a dependence on nucleotide concentration and had maximal values very similar to the equivalent parameters observed with ATP. On the basis of these fluorescence data, Bagshaw et  To test this view directly, it would be necessary to compare the rate of irreversible ATP binding and the maximum rate of the fluorescence change at 2O"C, 0.1 M KCl, pH = 7, the conditions used by Bagshaw et al. (8). Unfortunately, as discussed under "Results," this is not possible to accomplish experimentally at the present time. However, we were able to perform this experiment under the not too dissimilar conditions of 15"C, 0.1 M KCl, pH = 6.4 (Fig. 8) and here our results strongly suggest that the rate of irreversible ATP binding is, indeed, considerably faster than the maximal rate of the fluorescence enhancement. It, therefore, appears that, for ATP, the maximal rate of the fluorescence enhancement represents the rate of the initial P; burst (k3 + k-s), while for ADP and ATPyS it represents the rate of the conformational change, kz.
Another observation which we consistently made in regard to both the irreversible ATP binding and the initial Pi burst was that the last 20 to 30% of the reaction appeared to occur at a somewhat lower rate. If this is due to a small fraction of S-l which still binds ATP irreversibly but at a somewhat lower rate, it might partially account for the unexpectedly high values for the K,,, of the rabbit myosin ATPase observed by Taylor (0.25 pM) (5) and Taylor and Weeds (2.6 pM) (7).
On the basis of Scheme 1, K, = k3k4/(k3 + kma)Klkz (8). Therefore, if K,kz were low for some of the S-l, it might tend to increase the observed value of K,,,. Further work will be required to determine if this is indeed the explanation for the observation that the K,,, of the myosin ATPase is about 1 order of magnitude higher than expected on the basis of Scheme 1.
Our finding that the fluorescence increase is mainly associated with the initial P, burst rather than the binding of ATP is of interest in regard to the mechanism of the actomyosin ATPase. Both Chock et al. (14) and Sleep and Taylor (12) observed that a fluorescence increase occurred following the dissociation of acto-S-l by ATP. Since Taylor had suggested that no fluorescence change was associated with the initial Pi burst, Sleep and Taylor (12)  On the other hand, Chock et al. (14) suggested that the fluorescence increase which occurs following dissociation of the acto-S-l by ATP was due to the initial Pi burst: AM+ TsAM.T$M*.T+AeM**.D.P,+A The demonstration in this paper that a marked increase in fluorescence is associated with the initial Pi burst strongly supports the mechanism of Chock et al. (14) and makes it unnecessary to postulate the state Mt -T.
The data presented in this paper clarify one other feature of the actomyosin ATPase mechanism. The results of Chock et al. (14) and Sleep and Taylor (12) showed that the rate of dissociation of AM by ATP, i.e. the rate of formation of M* . Tin Scheme 10, was very rapid. In fact, this rate appeared to be much faster than the rate of M* . T formation from M + T since the maximal rate of the fluorescence increase was relatively slow with S-l alone and this rate was thought to be the maximal rate of M* . T formation in the absence of actin. However, the data presented in this paper suggest that k2, the maximal rate of formation of M* . T from M + T, may in fact be as fast as the rate of formation of M*. T from AM + T.
Thus, it may not be necessary to postulate that actin affects the maximal rate of formation of M* . T. Table I summarizes under varied conditions our values for the second order binding constant of ATP to myosin (Klk2) and the rate of the initial P, burst, i.e. the maximal rate of the fluorescence change (k3 + km3). As can be seen, KlkY is relatively insensitive to changes in temperature or pH but does tend to decrease markedly as the ionic strength is increased.
On the other hand, KS + km3 increases markedly with increasing temperature or pH and, to a lesser extent, with increasing ionic strength. One of the key questions about the mechanism of the actomyosin ATPase is the nature of the rate-limiting step, that is whether the initial Pi burst is identical to the ratelimiting transition from the refractory to the nonrefractory state (14). Obtaining the rate of the initial Pi burst from the maximal rate of the fluorescence increase should be very helpful in solving this problem. In this regard, preliminary data (21) suggest that at 15"C, the rate of the initial Pi burst is considerably faster than the maximum actin-activated ATPase rate. Therefore, at least at 15O"C, the initial Pi burst does not appear to be the rate-limiting step in the actomyosin ATPase cycle.