Rate of phosphate release after photoliberation of adenosine 5'-triphosphate in slow and fast skeletal muscle fibers.

Inorganic phosphate (Pi) release was determined by means of a fluorescent Pi-probe in single permeabilized rabbit soleus and psoas muscle fibers. Measurements of Pi release followed photoliberation of approximately 1.5 mM ATP by flash photolysis of NPE-caged ATP in the absence and presence of Ca2+ at 15 degrees C. In the absence of Ca2+, Pi release occurred with a slow rate of 11 +/- 3 microM . s-1 (n = 3) in soleus fibers and 23 +/- 1 microM . s-1 (n = 10) in psoas fibers. At saturating Ca2+ concentrations (pCa 4.5), photoliberation of ATP was followed by rapid force development. The initial rate of Pi release was 0.57 +/- 0.05 mM . s-1 in soleus (n = 13) and 4.7 +/- 0.2 mM . s-1 in psoas (n = 23), corresponding to a rate of Pi release per myosin head of 3.8 s-1 in soleus and 31.5 s-1 in psoas. Pi release declined at a rate of 0.48 s-1 in soleus and of 5.2 s-1 in psoas. Pi release in soleus was slightly faster in the presence of an ATP regenerating system but slower when 0.5 mM ADP was added. The reduction in the rate of Pi release results from an initial redistribution of cross-bridges over different states and a subsequent ADP-sensitive slowing of cross-bridge detachment.     

Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1.

The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.     

Reactions of the sarcoplasmic reticulum calcium adenosinetriphosphatase with adenosine 5'-triphosphate and Ca2+ that are not satisfactorily described by an E1-E2 model

Phosphorylation of the sarcoplasmic reticulum calcium ATPase, E, is first order with kb = 70 +/- 7 s-1 after free enzyme was mixed with saturating ATP and 50 microM Ca2+; this is one-third the rate constant of 220 s-1 for phosphorylation of enzyme preincubated with calcium, cE.Ca2, after being mixed with ATP under the same conditions (pH 7.0, Ca2+-loaded vesicles, 100 mM KCl, 5 mM Mg2+, 25 degrees C). Phosphorylation of E with ATP and Ca2+ in the presence of 0.25 mM ADP gives approximately 50% E approximately P.Ca2 with kobsd = 77 s-1, not the sum of the forward and reverse rate constants, kobsd = kf + kr = 140 s-1, that is expected for approach to equilibrium if phosphorylation were rate limiting. These results show that (1) kb represents a slow conformational change, rather than phosphoryl transfer, and (2) different pathways are followed for the phosphorylation of E and of cE.Ca2. The absence of a lag for phosphorylation of E with saturating ATP and Ca2+ indicates that all other steps, including the binding of Ca2+ ions and phosphoryl transfer, have rate constants of greater than 500 s-1. Chase experiments with unlabeled ATP or with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) show that the rate constants for dissociation of [gamma-32P]ATP and Ca2+ are comparable to kb. Dissociation of ATP occurs at 47 s-1 from E.ATP.Ca2+ and at 24 s-1 from E.ATP. Approximately 20% phosphorylation occurs following an EGTA chase 4.5 ms after the addition of 300 microM ATP and 50 microM Ca2+ to enzyme. This shows that Ca2+ binds rapidly to the free enzyme, from outside the vesicle, before the conformational change (kb). The fraction of Ca2+-free E.[gamma-32P]ATP that is trapped to give labeled phosphoenzyme after the addition of Ca2+ and a chase of unlabeled ATP is half-maximal at 6.8 microM Ca2+, with a Hill slope of n = 1.8. The calculated dissociation constant for Ca2+ from E.ATP.Ca2 is approximately 2.2 X 10(-10) M2 (K0.5 = 15 microM). The rate constant for the slow phase of the biphasic reaction of E approximately P.Ca2 with 1.1 mM ADP increases 2.5-fold when [Ca2+] is decreased from 50 microM to 10 nM, with half-maximal increase at 1.7 microM Ca2+. This shows that Ca2+ is dissociating from a different species, aE.ATP.Ca2, that is active for catalysis of phosphoryl transfer, has a high affinity for Ca2+, and dissociates Ca2+ with k less than or equal to 45 s-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Time-resolved measurements of phosphate release by cycling cross-bridges in portal vein smooth muscle.

The rate of release of inorganic phosphate (Pi) from cycling cross-bridges in rabbit portal-anterior mesenteric vein smooth muscle was determined by following the fluorescence of the Pi-reporter, MDCC-PBP (Brune, M., J. L. Hunter, S. A. Howell, S. R. Martin, T. L. Hazlett, J. E. T. Corrie, and M. R. Webb. 1998. Biochemistry. 37:10370-10380). Cross-bridge cycling was initiated by photolytic release of ATP from caged-ATP in Triton-permeabilized smooth muscles in rigor. When the regulatory myosin light chains (MLC20) had been thiophosphorylated, the rate of Pi release was biphasic with an initial rate of 80 microM s-1 and amplitude 108 microM, decreasing to 13.7 microM s-1. These rates correspond to fast and slow turnovers of 1.8 s-1 and 0.3 s-1, assuming 84% thiophosphorylation of 52 microM myosin heads. Activation by Ca2+-dependent phosphorylation subsequent to ATP release resulted in slower Pi release, paralleling the rate of contraction that was also slower than after thiophosphorylation, and was also biphasic: 51 microM s-1 and 13.2 microM s-1. These rates suggest that the activity of myosin light chain kinase and phosphatase ("pseudo-ATPase") contributes <20% of the ATP usage during cross-bridge cycling. The extracellular "ecto-nucleotidase" activity was reduced eightfold by permeabilization, conditions in which the ecto-ADPase was 17% of the ecto-ATPase. Nevertheless, the remaining ecto-ATPase activity reduced the precision of the estimate of cross-bridge ATPase. We conclude that the transition from fast to slow ATPase rates reflects the properties and forces directly acting on cross-bridges, rather than the result of a time-dependent decrease in activation (MLC20 phosphorylation) occurring in intact smooth muscle. The mechanisms of slowing may include the effect of positive strain on cross-bridges, inhibition of the cycling rate by high affinity Mg-ADP binding, and associated state hydrolysis.     

Dynamic interaction between actin and myosin subfragment 1 in the presence of ADP

The equilibrium and dynamics of the interaction between actin, myosin subfragment 1 (S1), and ADP have been investigated by using actin which has been covalently labeled at Cys-374 with a pyrene group. The results are consistent with actin binding to S1.ADP (M.D) in a two-step reaction, A + M.D K1 equilibrium A-M.D K2 equilibrium A.M.D, in which the pyrene fluorescence only monitors the second step. In this model, K1 = 2.3 X 10(4) M-1 (k+1 = 4.6 X 10(4) M-1 s-1) and K2 = 10 (k+2 less than or equal to 4 s-1); i.e., both steps are relatively slow compared to the maximum turnover of the ATPase reaction. ADP dissociates from both M.D and A-M.D at 2 s-1 and from A.M.D at greater than or equal to 500 s-1; therefore, actin only accelerates the release of product from the A.M.D state. This model is consistent with the actomyosin ATPase model proposed by Geeves et al. [(1984) J. Muscle Res. Cell Motil. 5, 351]. The results suggest that A-M.D cannot break down at a rate greater than 4 s-1 by dissociation of ADP, by dissociation of actin, or by isomerizing to A.M.D. It is therefore unlikely to be significantly occupied in a rapidly contracting muscle, but it may have a role in a muscle contracting against a load where the ATPase rate is markedly inhibited. Under these conditions, this complex may have a role in maintaining tension with a low ATP turnover rate.     

Energetics of the calcium-transporting ATPase

A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.     

Thermodynamics of the hydrolysis of adenosine 5'-triphosphate to adenosine 5'-diphosphate

The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic phosphate has been investigated using heat-conduction microcalorimetry. Enthalpies of reaction were measured as a function of ionic strength (0.05-0.66 mol kg-1), pH (6.4-8.8), and temperature (25-37 degrees C) in Tris/HCl buffer. The measured enthalpies were adjusted for the effects of proton ionization and metal ion binding, protonation and interaction with the Tris buffer, and ionic strength effects to obtain a value of delta H0 = -20.5 +/- 0.4 kJ mol-1 at 25 degrees C for the process, ATP4-(aq) + H2O(l) = ADP3-(aq) + HPO2-4(aq) + H+(aq) where aq is aqueous and l is liquid. Heat measurements carried out at different temperatures lead to a value of delta C0p = -237 +/- 30 J mol-1 K-1 for the above process.     

Early steps of the Mg(2+)-ATPase of relaxed myofibrils. A comparison with Ca(2+)-activated myofibrils and myosin subfragment 1.

The early steps of the Mg(2+)-ATPase activity of relaxed rabbit psoas myofibrils were studied in a buffer of near-physiological ionic strength at 4 degrees C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase, and the cleavage and release of product steps by the Pi burst method. The data obtained were interpreted by [formula: see text] where M represents the myosin heads with or without actin interaction. This work is a continuation of our study on Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. Here the constants obtained with relaxed myofibrils were compared with those with activated myofibrils and myosin subfragment 1 (S1). We find that whereas Ca2+ increases 80X the release of products (k4), it has little effect upon the kinetics of the initial binding and cleavage steps. As with activated myofibrils and S1, the second-order binding constant for ATP (k2/K1) was about 1 microM-1 s-1 and the ATP was bound very tightly. With activated myofibrils, it was difficult to obtain an estimate for the koff for ATP(k-2) but it is much less than kcat. Here with relaxed myofibrils we estimate k-2 less than 8 x 10(-4) s-1, which is considerably smaller than kcat (0.019 s-1) and also previous estimates for this constant. The overall Kd for ATP to relaxed myofibrils is less than 8 x 10(-10) M. With S1 this Kd is about 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of ATP and inorganic phosphate release during hydrolysis of ATP by rabbit skeletal actomyosin subfragment 1. Oxygen exchange between water and ATP or phosphate

We have used the technique of phosphate: water oxygen exchange to measure the rate of ATP and Pi release and Pi binding to myosin subfragment 1 and actomyosin subfragment 1 from rabbit skeletal muscle. The oxygen exchange distributions for ATP and Pi release fit a simple kinetic model with a single set of rate constants for each step. For actomyosin subfragment 1 (20 degrees C, pH 7.0, I = 50 mM), the rate constant governing ATP release is approximately 8 s-1, Pi release is at approximately 60 s-1 and Pi rebinds to an ADP state at greater than 120 M-1 s-1. These rate constants are similar to those that may occur for undistorted cross-bridges within glycerinated rabbit psoas fibers (Bowater, R., Webb, M. R., and Ferenczi, M. A. (1989) J. Biol. Chem. 264, 7193-7201.     

Rate-limiting step in the actomyosin adenosinetriphosphatase cycle: studies with myosin subfragment 1 cross-linked to actin

Although there is agreement that actomyosin can hydrolyze ATP without dissociation of the actin from myosin, there is still controversy about the nature of the rate-limiting step in the ATPase cycle. Two models, which differ in their rate-limiting step, can account for the kinetic data. In the four-state model, which has four states containing bound ATP or ADP . Pi, the rate-limiting step is ATP hydrolysis (A . M . ATP in equilibrium A . M . ADP . Pi). In the six-state model, which we previously proposed, the rate-limiting step is a conformational change which occurs before Pi release but after ATP hydrolysis. A difference between these models is that only the four-state model predicts that almost no acto-subfragment 1 (S-1) . ADP . Pi complex will be formed when ATP is mixed with acto . S-1. In the present study, we determined the amount of acto . S-1 . ADP . Pi formed when ATP is mixed with S-1 cross-linked to actin [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306]. The amount of acto . S-1 . ADP . Pi was determined both from intrinsic fluorescence enhancement and from direct measurement of Pi. We found that at mu = 0.013 M, the fluorescence magnitude in the presence of ATP of the cross-linked actin . S-1 preparation was about 50% of the value obtained with S-1, while at mu = 0.053 M the fluorescence magnitude was about 70% of that obtained with S-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rate of ATP synthesis by dynein

The rates of ATP synthesis and release by the dynein ATPase were determined in order to estimate thermodynamic parameters according to the pathway: (Formula: see text). Dynein was incubated with high concentrations of ADP and Pi to drive the net synthesis of ATP, and the rate of ATP production was monitored fluorometrically by production of NADPH through a coupled assay using hexokinase and glucose-6-phosphate dehydrogenase. The turnover number for the rate of release of ATP from 22S dynein was 0.01 s-1 per site at pH 7.0, 28 degrees C, assuming a molecular weight of 750 000 per site. The same method gave a rate of ATP synthesis by myosin subfragment 1 of 3.4 X 10(-4) s-1 at pH 7.0, 28 degrees C. The rate of ATP synthesis at the active site was estimated from the time dependence of medium phosphate-water oxygen exchange. Dynein was incubated with ADP and [18O] Pi, and the rate of loss of the labeled oxygen to water was monitored by 31P NMR. A partition coefficient of 0.31 was determined, which is equal to k-2/(k-2 + k3). Assuming k3 = 8 s-1 [Johnson, K.A. (1983) J. Biol. Chem. 258, 13825-13832], k-2 = 3.5 s-1. From the rates of ATP binding and hydrolysis measured previously (Johnson, 1983), the equilibrium constants for ATP binding and hydrolysis could be calculated: K1 = 5 X 10(7) M-1 and K2 = 14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and hydrolysis of ATP by cardiac myosin subfragment 1: effect of solution parameters on transient kinetics

Transient kinetic data of ATP binding and cleavage by cardiac myosin subfragment 1 (S1) were obtained by fluorescence stopped flow and analyzed by using computer modeling based on a consecutive, reversible two-step mechanism: (formula: see text) where M1 and M12 denote myosin species with enhanced fluorescence and K'O = K0/(K0[ATP] + 1). The kinetic constants K0, k12, k23, and k32 and the fractional contributions of M1 and M12 to the total fluorescence are analyzed over a range of systematically varied solution parameters. The initial ATP binding equilibrium (K0), which decreases with increasing pH, is facilitated by a positively charged protein residue with a pK of 7.1. An active-site charge of +1.5 is determined from the ionic strength dependence. The rate constants k12, k23, and k32 also exhibit pK's near neutrality but increase with increasing pH. The majority of the large (-54 kJ/mol) negative free energy of ATP binding occurs upon S1 isomerization, k12, and a large increase in entropy (183 J/kmol at 15 degrees C) is associated with the cleavage step. The equilibrium constant for the cleavage step, K2, is determined as 3.5 at pH 7.0, 15 degrees C, and 200 mM ionic strength. There are no significant changes in fractional contributions to total fluorescence enhancement due to solvent-dependent conformational changes of S1 in these data. When values for the combined rate constants are calculated and compared with those determined by graphical analysis, it is observed that graphical analysis overestimates the binding rate constant (K0k12) by 25% and the hydrolysis rate constant (k23 + k32) by as much as 30%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPases. An example of an uncompetitive inhibitor.

2,3-Butanedione monoxime (BDM) reversibly inhibits force production in muscle. At least part of its action appears to be directly on the contractile apparatus. To understand better its mechanism of action, we studied the effect of BDM on the steps of myosin subfragment 1 Mg(2+)-ATPase in 0.1 M potassium acetate, pH 7.4. Because of the rapidity of certain processes, we experimented at 4 degrees C and our main technique was the rapid flow quench method. By varying the experimental conditions (relative concentrations of reagents, time scale, quenching agent), it was possible to study selectively the different steps of the S1 Mg(2+)-ATPase: [formula: see text] At saturation (20 mM), BDM had two major effects on the ATPase. First, it increased the equilibrium constant of the cleavage step (K3) from 2 to > 10. Second, it slowed the kinetics of the release of Pi by an order of magnitude (k4; from 0.054 to 0.004 s-1). By contrast, the kinetics of the binding of ATP (k) and the release of ADP (k6) were little affected by BDM. Thus, the oxime appears to interact specifically with M**.ADP.Pi, and it is a rare example of an uncompetitive inhibitor. Its effect is to reduce the steady-state concentration of the "strong" actin binding state M*.ADP and to increase that of the "weak" binding state, M**.ADP.Pi. The effect of BDM on the initial ATPase of Ca2+ activated myofibrils was very similar to that on S1 ATPase. Thus, with myofibrils too BDM seems to exert its main effect subsequent to the initial binding and cleavage steps.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism for nucleotide exchange in monomeric actin

Rabbit skeletal muscle G-actin has been treated to obtain ADP, 1,N6-ethenoadenosine diphosphate (epsilon-ADP), or 1,N6-ethenoadenosine triphosphate (epsilon-ATP) at the nucleotide binding site and either Mg2+ or Ca2+ at high- and moderate-affinity metal binding sites. Apparent rates or rate constants for the displacement of the actin-bound nucleotides by epsilon-ATP or ATP have been obtained by stopped-flow measurements at pH 8 and 20 degrees C of the fluorescence difference between bound and free epsilon-ATP or epsilon-ADP. In the presence of Ca2+, displacement of ADP by epsilon-ATP or epsilon-ADP by ATP is a biphasic process, but in the presence of low (less than 10 microM) Mg2+ concentrations, it is a slow first-order process. At high levels of Mg2+ (greater than 50 microM), low ADP concentrations displace epsilon-ATP from G-actin as a consequence of Mg2+ binding to moderate-affinity sites on the actin. Displacement of epsilon-ATP by ATP in the presence of either Ca2+ or Mg2+ is slow at low ATP concentrations, but the rate is increased by high ATP concentrations. Using ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we find that nucleotide exchange is affected differently by the removal of Ca2+ from the high-affinity site compared to Ca2+ removal from moderate-affinity sites. A mechanism for the displacement reaction is proposed in which there are two forms of an actin-ADP complex and metal binding influences the ratio of these forms as well as the binding of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryoenzymic studies on actomyosin ATPase: kinetic evidence for communication between the actin and ATP sites on myosin.

The post-ATP binding steps of myosin subfragment 1 (S1) and actomyosin subfragment 1 (actoS1) ATPases were studied at -15 degrees C with 40% ethylene glycol as antifreeze. The cleavage and release of Pi steps were studied by the rapid-flow quench method and the interaction of actin with S1 plus ATP by light scattering in a stopped-flow apparatus. At -15 degrees C, the interaction of actin with S1 remains tight, and the Km for the activation of S1 ATPase is very small (0.3 microM). The chemical data were interpreted by E + ATP----E*.ATP----E**.ADP.Pi----E*.ADP----products, where E is S1 or actoS1. In Pi burst experiments with S1, there was a large Pi burst of free Pi, but E**.ADP.Pi could not be detected. Here the predominant complex in the seconds time range is E*.ATP and in the steady-state E*.ADP. With actoS1, there was a small Pi burst of E**.ADP.Pi, evidence that the cleavage steps for S1 and actoS1 are different. From the stopped-flow experiments, the dissociation of actoS1 by ATP was complete, even at actin concentrations 60X its Km. Further, no interaction of actin with the key intermediate M*.ATP could be detected. Therefore, at -15 degrees C, actoS1 ATPase occurs by a dissociative pathway; in particular, the cleavage step appears to occur in the absence of actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.     

The phosphorylation potential generated by respiring bovine heart submitochondrial particles.

A phosphorylation potential deltaGp, where deltaGp = deltaGo' + RT2.303 log ([ATP]/([ADP][Pi])), of approx. 44.3 kJ.mol-1 (10.6 kcal.mol-1) was generated by submitochondrial particles that were oxidizing either NADH or succinate. Addition of adenylyl imidodiphosphate, which should suppress adenosine triphosphatase activity of any uncoupled particles, did not raise the phosphorylation potential. Raising the Pi concentration slightly increased the magnitude of the value for [ATP]/[ADP], but this did not fully compensate for the increased Pi concentration, so that the phosphorylation potential decreased slightly as the Pi concentration was raised. The phosphorylation potential developed by submitochondrial particles is lower than that generated by phosphorylating membrane vesicles from some bacteria, and is also less than that developed externally by mitochondria, but is strikingly close to the phosphorylation potential that is generated internally by mitochondria.     

Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin.

Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.     

Reaction heats and heat capacity changes for intermediate steps of the ATP hydrolysis catalyzed by myosin subfragment 1

The interaction of myosin Subfragment 1 with ATP in 0.1 M KCl containing 0.01 M MgCl2 and 0.02 M Tris/HCl (pH 8.0) was studied by microcalorimetry at temperatures of 4, 12, and 23 degrees C so that values of the heat capacity change (delta Cp) could be obtained for intermediate steps of the ATPase cycle. The delta Cp values are large compared to the value for the overall cycle, indicating that large changes in the hydrophobic effect are involved in transitions between different intermediate states. However, the heat capacity changes themselves show peculiar temperature dependences. Thus bindings of ATP and ADP to Subfragment 1, both of which are strongly exothermic processes, take place with large negative delta Cp of about -3 kJK-1 mol-1 between 4 and 12 degrees C but with very small delta Cp of 0.3-0.4 kJ K-1 mol-1 between 12 and 23 degrees C. On the contrary, the delta Cp for the endothermic hydrolysis of ATP bound to Subfragment 1 is positive (congruent to kJK-1 mol-1) in the lower temperature range but strongly negative (congruent to -4 kJK-1 mol-1) in the higher temperature range. The magnitude of delta Cp for the slow Pi dissociation process is similar but its sign is just opposite to that for the hydrolysis. These anomalous changes in the heat capacity may be due to the temperature-induced changes in a balance between large opposing effects which result from distinct, local conformation changes within the Subfragment 1 molecule.     

Kinetic studies on the association and dissociation of myosin subfragment 1 and actin.

The reactions of pyrene-labeled actin with myosin subfragment 1 (S1) and S1-ligand complexes at low ionic strength are described by the schemes [formula: see text] where M refers to a myosin head; A is actin; L is ligand; the asterisk refers to a high fluorescence state of actin; and K1 and K3 are association constants. K1 is reduced approximately 10-fold for M.ADP or M.pyrophosphate versus M alone. The rate constant of the isomerization step (k2) is 150-200 s-1 for A*M, A*M.ADP, and A*M-pyrophosphate (20 degrees C). The interaction between the ligand the actin binding sites reduces K2 from 2,000 for A*M to 50-100 for A*M.ADP and to approximately unity for A*M-pyrophosphate. The A*M.ADP state is equated with the AM'.ADP state of Sleep and Hutton (Sleep, J., A., and Hutton, R. L. (1980) Biochemistry 19, 1276-1283).