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Query: UNIPROT:Q8N5D0 (ADP)
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A calorimetric titration method was used to study ADP binding to native myosin. Data were analyzed by assuming that the myosin molecule has n independent and identical sites for ADP binding. The enthalpy change (deltaH), the binding constant (K), and n were determined. In 0.5 M KCl, 0.01 M MgCl2, and 0.02 M Tris/HCl (pH 7.8), we found: at 0 degrees, deltaH = -57.1 +/- 3.2 kJ-mol-1, log K = 6.42 +/- 0.13, n = 1.49 +/- 0.07; at 12 degrees, deltaH = 73.1 +/- 3.2 kJ-mole-1, log K = 6.08 +/- 0.13, and n = 1.74 +/- 0.07. The average heat capacity change on ADP binding to myosin between 0 and 12 degrees is thus -1.4 +/- 0.4 kJ-mol-1-K-1. Reasonably consistent results were obtained at 25 degrees, suggesting ADP binding to myosin is as strongly exothermic as at lower temperatures, although further interpretation of this result seems unwarranted, mainly because of the instability of myosic at this temperature. The number of protons released on binding of ADP to myosin was determined in separate experiments. The value was 0.19 +/- 0.02 at both 0 and 12 degrees. The reaction of protons with Tris thus contributes about -9.5 kJ-mol-1 to the observed heat on ADP binding.
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PMID:Calorimetric studies of the interaction of myosin with ADP. 13 38

The gamma-phosphoryl groups of two intermediates (M-ATP and M-ADP-P1) in the pathway of MgATP hydrolysis by myosin undergo extensive oxygen exchange with water. Actin activates the overall rate of hydrolysis at a rate-limiting step which follows these exchange reactions. Thus, actin, by decreasing the turnover time of hydrolysis, would be expected to proportionately decrease the time available for oxygen exchange. Using subfragment 1 of myosin, the turnover time of hydrolysis can be varied over a wide range by changing the concentration of actin. An estimate for the rate constant of exchange can then be obtained by relating these turnover times to measured values for oxygen exchange (incorporation of 18O from H218O into the inorganic phosphate (Pi) released by hydrolysis). The results of such an experiment, with turnover times between 0.2 and 25 s, indicate that, for each gamma-phosphoryl group, one oxygen from the medium is added rapidly (to cleave the phosphoryl group or form a pentacoordinate phosphroyl complex); two more oxygens exchange with a rate constant, kc, of about 1 s-1; and a fourth oxygen exchanges slowly with ke about 0.2 s-1. The higher value is about 18 times smaller than the rate constant, 5-3, for the reverse cleavage step of the myosin pathway, which is postulated to be responsible for oxygen exchange. The data, then, indicate that the rate-limiting step for oxygen exchange is not k-3, but may be the rate of rotation of oxygens around the phosphorus atom, with one oxygen severely restricted by its binding to the active site. The finding that kc differs for the four oxygens in each phosphate group is related to past observations on myosin-catalyzed oxygen exchange.
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PMID:Mechanism of oxygen exchange in actin-activated hydrolysis of adenosine triphosphate by myosin subfragment 1. 13 40

During Mn(II)-ATP hydrolysis by myosin, the predominant intermediate formed at the burst site of the enzyme below 10 degrees is the myosin-ADP complex formed by adding ADP to myosin, while above 10 degrees it is the myosin -ADP-P1 complex generated by ATP hydroolysis (Yazawa, Morita, & Yagi (1973) J. Biochem. 74, 1107; Hozumi & Tawada (1975) Biochim. Biophys. Acta 376, 1; Tawada & Yoshida (1975) J. Biochem. 78, 293). It is suggested that the second (non-burst) site of myosin predominantly forms the myosin-ATP complex (Hozumi & Tawada, ibid.). From these findings, it is expected that (i) myosin subfragment 1 (S1) having the burst site is bound to actin in Mn(II)-ATP solution containing ADP below 10 degrees, because it forms the S1-ADP complex even in the presence of ATP; (ii) the other S1, i.e., that having the non-burst site, is dissociated from actin, because it forms the S1-ATP complex. These two expectations were confirmed by viscosity measurements of acto-S1 solutions, giving a basis for the separation of S1 into two fractions: one having the burst site and the other having the non-burst site. S1 having the non-burst site could be extracted from partially papain [EC 3.4.22.2]-digested myofibrils of rabbit skeletal muscle with a solution containing MnCl2, ATP, and ADP at 0 degrees. S1 having the burst site was extracted from myofibrils already used for the extraction of S1 having the non-burst site, with a solution containing MgCl2 and ATP at 20 degrees. The former S1 fraction had Mg-ATPase [EC 3.6.1.3] activity, but scarcely showed any initial burst of Pi liberation. The latter S1 showed a Pi burst of more than 0.5 (M/M). The steady state ATPase activity of the former S1 was slightly higher than that of the latter. The burst size of normal S1, i.e., that extracted from papain-digested myofibrils with Mg-PPi or Mg-ATP, was 0.5 (M/M). The ultraviolet absorption spectrum of the non-burst type S1 was not changed by ADP but was changed by ATP, though the difference spectrum was distinct from that of normal S1 and the difference molar extinction coefficient at 289 nm was only 20% of that of normal S1. No significant difference was seen in the compositions of these two S1's and normal S1, as determined by SDS gel electrophoresis.
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PMID:Separation of myosin subfragment 1 into two fractions, one having the burst site and the other having the non-burst site. 13 98

beta-Naphthoquinone-4-sulfonate was used for chemical modification of amino groups of myosin. The reagent was found to affect also the sulfhydryl groups if the reaction was not prevented by previous disulfide exchange with cystamine. When cystamine protection was employed the ATPase (ATP phosphohydrolase, EC3.6.1.3) activity was enhanced in the presence of Mg2+ and decreased in the presence of K+ or Ca2+, a pattern typical of myosin with blocked essential amino groups. On addition of ATP or ADP a blueshift was observed in the fluorescent emission spectrum of beta-naphthoquinone-4-sulfonate bound by myosin, presumably owing to conformational changes in the environment of essential amino groups induced by the binding of nucleotides.
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PMID:Studies on the amino groups of myosin ATPase. III. Effect of nucleotides on the fluorescence of beta-naphthoquinone-4-sulfonate bound to amino groups of myosin. 13 47

1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis. 2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1 M KCl was measured in 0.5 M KCl, and found to be 0.93 mole/mole of myosin. 3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils. 4. The maximum amount of ADP bound to myofibrils in 0.5 M KCl was about 1.9 mole/mole of myosin. When about one mole of ADP was found to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was found per mole of myosin.
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PMID:Structure and function of the two heads of the myosin molecule. I. Binding of adenosine diphosphate to myofibrils during the adenosinetriphosphatase reaction. 13 77

Bovine cardiac myosin ATPase activity was rapidly inactivated by the purine disulfide analog of ATP,6,6'-dithiobis(inosinyl imidodiphosphate). Kinetic investigations showed that this analog acted as a site-specific reagent at 0 degrees with a Ki of 130 muM and a half-life of 8.2 min at saturating inhibitor concentrations. Concentrations (50 to 500 muM) of ATP, adenyl-5'-yl imidodiphosphate (AMP-PNP), or ADP that saturated the active site caused an enhancement in the rate of inactivation, indicating the purine disulfide analog was not reacting at the active site. Under these conditions saturation kinetic data were still observed with Ki values remaining unchanged (120 muM) but with the half-life of inactivation decreasing to 6.0 min (ATP) and 4.6 min (AMP-PNP) at saturating inhibitor concentrations. At concentrations greater than 0.5 mM ATP, AMP-PNP, or ADP there was a decrease in the rate of inactivation, implying protection by these nucleotides. However, saturation kinetics of inactivation could no longer be demonstrated, implying a change in the mechanism of inactivation. A comparison of the inactivation of the Mg2+, Ca2+, and EDTA-ATPase activities of cardiac myosin after modification by the purine disulfide analog showed that the Mg2+- and Ca2+ATPase activities plateaued at approximately 60% and 40%, respectively, while the EDTA-ATPase activity continued to decrease to below 10%. This evidence supports the suggestion that the purine disulfide analog was not reacting at the active site. Equilibrium dialysis experiments were used to measure the binding of [8-3H]AMP-PNP to native cardiac myosin, the thiopurine nucleotide-modified myosin, and the derivative formed by displacing the thiopurine nucleotide by cyanide (thiocyanato-myosin). Native myosin bound a total of 2.1 mol of AMP-PNP with a binding constant of 6.0 X 10(6) M-1. There was a 15 to 40% decrease in the number of AMP-PNP binding sites in the enzyme derivatives, but the active sites appeared not to be blocked since the association constants remained essentially unchanged (KA=3.9 X 10(6) M-1 for thiopurine nucleotide-myosin and 12.0 X 10(6) M-1 for thiocyanato-myosin). The kinetic studies and the binding experiments indicate that the purine disulfide analog reacts at a specific site other than the active site but do not offer support to earlier suggestions from skeletal myosin studies that this site is a possible ATP control site.
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PMID:Reaction of cardiac myosin with a purine disulfide analog of adenosine triphosphate. I. Kinetics of inactivation and binding of adenylyl imidodiphosphate. 13 83

The UV absorption difference spectrum of heavy meromyosin induced by adenylyl imidodiphosphate (AMP-PNP) was found to be changed by temperature. At higher temperatures, the shape of the difference spectrum resembled the ATP-form of difference spectrum induced by ATP. At lower temperatures, a different shape was observed, resembling that induced by ADP. This temperature transition was found in the presence of both MgCl2 and MnCl2. The transition temperatures, were 21 degrees and 9 degrees in the presence of MnCl2 and MgCl2, respectively. A similar temperature dependence was observed with the difference spectrum induced by ATP at the steady state. The transition temperatures in this case were 11 degrees and 4.5 degrees in the presence of MnCl2 and MgCl2, respectively. The similarity of the effects of the two kinds of divalent cation on both transitions indicates that the temperature induced transition between two species of heavy meromyosin-AMP-PNP complex mimics the step in APTase [EC 3.6.1.3] reaction in which the intermediate complex showing the ATP-form of difference spectrum changes to that showing the ADP-form. The equilibrium constant of the decay step of the ATP-form of difference spectrum to the ADP-form in ATPase is, therefore, thought to be highly temperature dependent. Thermodynamic parameters were calculated for the transition between the two species of heavy meromyosin AMP-PNP complex. Large decreases in enthalpy and entropy were observed, while the standard free energy change was small. The results suggest that the intermediate showing the ATP-form of difference spectrum hardly changes to the forward direction in the ATPase reaction at higher temperature. The complex appears to be so stable in the steady state that almost all the myosin is present as this complex. The decay step in ATPase of the difference spectrum from the ATP-form to to the ADP-form may be coupled to muscular contraction. The temperature induced transition of heavy meromyosin AMP-PNP complex may, therefore, provide information concerning the state of myosin in active muscles.
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PMID:Temperature induced analog reaction of adenylyl imidodiphosphate to an intermediate step of heavy meromyosin adenosine triphosphatase. 13 5

In the course of MG-dependent ATP splitting by heart actomyosin, an "energy rich" actomyosin-ADP complex is formed, which promotes the incorporation of phosphate 32P into ATP in myofibrils. The rate of this ATP-phosphate exchange reaction depends on the extent of actin-myosin overlap which can be decreased by stretching glycerinated muscle fibres. In heart muscle, the calcium-ion dependence of this reaction is similar to that of the actomyosin ATPase, the tension, and "immediate fibre stiffness" (which is "hookean" and which is a measure for the number of myosin cross-bridges attached to and interacting with actin). These findings suggest that calcium increases the amount of "contractile" actomyosin-ADP complexes. The proportionality between tension and ATPase activity further suggests that the rate-limiting step of the cross-bridge cycle (which determines the molecular turnover number, the "Wechselzahl" of the ATPase) is only little affected by calcium ions. These ions act by recruiting more bridges rather than by accelerating their reactions. In addition, the depressing effect of inorganic phosphate on the contractile tension and its presumable role in energetic insuffciency will be discussed.
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PMID:Ca++ activation of ATPase activity, ATP-Pi exchange, and tension in briefly glycerinated heart muscle. 14 Jun 54

A considerable blue shift was observed in the absorption spectrum of the trinitrophenyl moiety attached to a functional epsilon-lysyl amino group of subfragment-1, heavy meromyosin and myosin on addition of ATP or ATP analogs. The resulting difference spectra showed a maximum at 320 and a minimum at 365 nm. The greatest spectral change was observed with a non-hydrolyzable ATP analog, adenosine 5'-(beta,gamma-imino)triphosphate and it decreased in the order adenosine 5'-(beta,gamma-imino)triphosphate, ATP and ADP. The ATP-induced difference spectrum changed to that of ADP upon the hydrolysis of ATP. The observed spectra were depended on temperature and ionic strength. Difference spectra were produced also by ITP, IDP and pyrophosphate while AMP was practically ineffective. Mg2+ also caused small spectral changes which are not identical with those induced by ATP analogs. On the basis of measurements carried out on a model compound, it is assumed that as a consequence of the reaction of ATP with a myosin head, the environment of the functional lysyl residue becomes less polar, i.e. it becomes buried in the hydrophobic core of the molecule. Changes on addition of ATP or its analogs were observed also in the circular dichroic (CD) spectrum of trinitrophenylated subfragment-1, which also points to conformational changes in the vicinity of the functional lysyl residue.
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PMID:Studies on the amino groups of myosin ATPase. IV. Effects of ATP and its analogs on the spectral properties of trinitrophenylated myosin and its active fragments. 14 48

The extent of ATP synthesis from ADP and Pi at the active centre of myosin subfragment 1 has been reinvestigated. The results have been interpreted using a treatment which is not dependent on the number or nature of the intermediates in the ATPase mechanism. An average value for the binding constant of ATP of (3.25 +/- 0.96) X 10(11) M-1 at pH 8.0 23 degrees C and ionic strength 0.12 M was obtained. Additional evidence is given to confirm that synthesis at the active site has been investigated.
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PMID:The binding constant of ATP to myosin S1 fragment. 14 53


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