The reversibility of adenosine triphosphate cleavage by myosin

For the simplest kinetic model the reverse rate constants (k₋₁ and k₋₂) associated with ATP binding and cleavage on purified heavy meromyosin and heavy meromyosin subfragment 1 from rabbit skeletal muscle in the presence of 5mm-MgCl₂, 50mm-KCl and 20mm-Tris–HCl buffer at pH8.0 and 22°C are: k₋₁<0.02s⁻¹ and k₋₁=16s⁻¹. Apparently, higher values of k₋₁ and k₋₂ are found with less-purified protein preparations. The values of k₋₁ and k₋₂ satisfy conditions required by previous ¹⁸O-incorporation studies of H₂¹⁸O into the P_i moiety on ATP hydrolysis and suggest that the cleavage step does involve hydrolysis of ATP or formation of an adduct between ATP and water. The equilibrium constant for the cleavage step at the myosin active site is 9. If the cycle of events during muscle contraction is described by the model proposed by Lymn & Taylor (1971), the fact that there is only a small negative standard free-energy change for the cleavage step is advantageous for efficient chemical to mechanical energy exchange during muscle contraction.     

Synthesis of adenosine triphosphate and exchange between inorganic phosphate and adenosine triphosphate in sodium and potassium ion transport adenosine triphosphatase.

Radioactive adenosine triphosphate was synthesized transiently from adenosine diphosphate and radioactive inorganic phosphate by sodium and potassium adenosine triphosphatase from guinea pig kidney. In a first step, K+-sensitive phosphoenzyme was formed from radioactive inorganic phosphate in the presence of magnesium ion and 16 mM sodium ion. In a second step the addition to the phosphoenzyme of adenosine diphosphate with a higher concentration of sodium ion produced adenosine triphosphate. Recovery of adenosine triphosphate from the phosphoenzyme was 10 to 100% in the presence of 96 to 1200 mM sodium ion, respectively. Potassium ion (16mM) inhibited synthesis if added before or simultaneously with the high concentration of sodium ion but had no effect afterward. The half-maximal concentration for adenosine diphosphate was about 12 muM. Ouabain inhibited synthesis. The ionophore gramicidin had no significant effect on the level of phosphoenzyme nor on the rate nor on the extent of synthesis of adenosine triphosphate. The detergent Lubrol WX reduced the rate of phosphoenzyme break-down and the rate of synthesis but did not affect the final recovery. Phospholipase A treatment inhibited synthesis. In a steady state, the enzyme catalzyed a slow ouabain-sensitive incorporation or inorganic phosphate into adenosine triphosphate. These results and other suggest that binding of sodium ion to a low affinity site on phosphoenzyme formed from inorganic phosphate is sufficient to induce a conformational change in the active center which permits transfer of the phosphate group to adenosine diphosphate.     

Quantitative analysis of intermediate phosphate-oxygen exchange during hydrolyses by ATPases

A simple mathematical model is presented that predicts the paradoxical effect of substrate concentration on the extent of oxygen exchange into the phosphate produced during hydrolyses of nucleotide triphosphates by ATPases in labelled water. The same model also predicts the deviations from Michaelis-Menten kinetics observed at low salt concentrations.     

Induction by nucleotide triphosphate hydrolysis of a form of sarcoplasmic reticulum ATPase capable of medium phosphate-oxygen exchange in presence of calcium.

Sarcoplasmic reticulum vesicles rendered leaky by exposure to alkaline pH, like intact vesicles, catalyze a rapid Mg2+-dependent exchange of oxygens of medium Pi with water. The exchange with 10 mM Pi is strongly inhibited by 0.15 mM Ca2+. Upon addition and hydrolysis of ITP or ATP, a rapid phosphate-oxygen exchange is observed even with 0.15 mM Ca2+ present and a definite but smaller exchange at 8 mM Ca2+. Oxygen exchange per Pi formed is greater with ITP than with ATP. When no Pi is initially present, the extent of oxygen exchange is increased with time of incubation as Pi is formed. With 18O-labeled Pi present, ATP hydrolysis accelerates 18O loss. The results show that much of the oxygen exchange occurs as a result of reversible binding of medium Pi. Thus the binding and cleavage of ITP or ATP overcomes the Ca2+ inhibition of the medium Pi in equilibrium HOH exchange. Such findings support the concept that the cleavage cycle includes a transient conformational form which can reversibly react with Pi to give a phosphoryl enzyme and resultant oxygen exchange or in a rate-limiting step decay to a form with high Ca2+ and NTP affinity.     

The reaction between actomyosin and adenosine triphosphate

1. A study is made of the effect of adenosine triphosphate (ATP) upon the viscosity of solutions of actomyosin in 0.5 M KCl. 2. The observed effects are discussed in terms of an initial drop of the viscosity (viscosity response) and its subsequent slow reversal (recovery effect). The latter is ascribed to a decrease in the ATP concentration through enzymatic hydrolysis. 3. The recovery effect is inhibited by Mg, activated by Ca, in accordance with the effect of these ions on the activity of myosin-ATPase. 4. The viscosity response is not inhibited, probably promoted by Mg. It is not promoted, probably inhibited by Ca. 5. The viscosity response is induced not only by ATP, but to a certain extent also by inosinetriphosphate, inorganic triphosphate, and inorganic pyrophosphate, not by adenosine diphosphate or monophosphate. 6. The viscosity response could be obtained with enzymatically inactive myosin. 7. It is concluded that the effect of ATP upon myosin does not depend on its enzymatic hydrolysis.     

Development of a charcoal paper adenosine triphosphate:pyrophosphate exchange assay: Kinetic characterization of NEDD8 activating enzyme

Ubiquitin activating enzyme (UAE, UBE1, or E1) and seven known homologous “E1s” initiate the conjugation pathways for ubiquitin and 16 other ubiquitin-like modifiers (ULMs) found in humans. The initial step catalyzed by E1s uses adenosine triphosphate (ATP) to adenylate the C terminus of the appropriate ULM and results in the production of inorganic pyrophosphate (PPi). The mechanism of these enzymes can be studied with assays that measure the rate of ULM-dependent ATP:PPi exchange. The traditional method follows the initial velocity of [³²P]PPi incorporation into ATP by capturing the nucleotide on activated charcoal powder to separate it from excess [³²P]PPi and then measuring [³²P]ATP in a scintillation counter. We have modified the method by using charcoal paper to capture the nucleotide and a phosphorimager to quantify the [³²P]ATP. The significant increase in throughput that these modifications provide is accomplished without any sacrifice in sensitivity or accuracy compared with the traditional method. To demonstrate this, we reproduce and extend the characterization of the NEDD8 activating enzyme.