The reversibility of adenosine triphosphate cleavage by myosin

Abstract

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-MgCl(2), 50mm-KCl and 20mm-Tris-HCl buffer at pH8.0 and 22 degrees 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 (18)O-incorporation studies of H(2) (18)O 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.

The Binding Constant of ATP to Myosin S1 Fragment

“…The results could be fitted by an exponential curve, with a rate constant of about 0.06 s-'. The expected rate of decay is equal to kcat, the turnover number of the ATPase, if synthesis takes place at the S1 active site, since bound ATP can only be removed from this site as products because kcat is much larger than k d i s s for ATP, as shown by Bagshaw and Trentham [4]. kcat was measured for the same S1 sample by following a single turnover of ATP using the enhancement of the tryptophan fluorescence [3] and found to be about 0.055 s-'.…”

“…The application of rapid kinetic techniques to the study of myosin ATPase established the basic characteristic features of the ATP hydrolysis, namely the relatively rapid cleavage of ATP on the enzyme, followed by the very slow release of products [1,2]. It was seen that the rate constant for ATP dissociation was much slower than for the cleavage step [3] and in fact much slower than the steady-state rate constant [4]. Using values for the rate constants for association, cleavage and rate-controlling step, which were obtained from these analyses, and taking the rate constant for ATP dissociation to be at least a factor of 10 lower than that for the rate-controlling step, an estimated value for K, of approximately 2-3 x M is obtained, which is in fair agreement with the value for K , determined experimentally at ATP Abbreviations.…”

The free energy for hydrolysis of a microtubule-bound nucleotide triphosphate is near zero: all of the free energy for hydrolysis is stored in the microtubule lattice [published erratum appears in J Cell Biol 1995 Apr;129(2):549]

“…Therefore, the standard free energy for forming the GMPCP-Pi-microtubule ternary complex would be +1.28 kcal (i.e., 2.18 -0.9), corresponding to an equilibrium constant equal to 0.114. For comparison, the free energy change is -1.3 kcal for conversion of the myosin-ATP to the myosin-ADP-Pi ternary complex (Bagshaw and Trentham, 1973).…”

“…The rate for tubulin-GMPCPP and tubulin-GMPCP subunit dissociation from microtubule ends were found to be about 0.65 and 128 s -1, respectively. The much faster rate for tubulin-GMPCP subunit dissociation provides direct evidence that microtubule dynamics can be regulated by nucleotide triphosphate hydrolysis.T HE hallmark for energy-transducing systems such as myosin (Bagshaw and Trentham, 1973), ion-transporting ATPases (Taniguchi, and Post, 1975;Pickart and Jencks, 1984), the mitochondrial ATPase (Grubmeyer et al, 1982) and chloroplast coupling factor (Feldman and Sigman, 1982) is a near-zero free energy for hydrolysis of bound nucleotide triphosphate (NTP)L Negligible free energy is released during NTP hydrolysis since this is stored in the protein conformation until useful work can be done. We have used the hydrolyzable GTP analogue GMPCPP, which contains a methylene linkage between the alpha and beta phosphates, to determine the free energy for hydrolysis of microtubule-bound NTP.…”

Sulphate is a competitive inhibitor of the binding of nucleotide to myosin A comparison with phosphate

“…Since k-2 is close to zero and /CZ * k, [6,7], by this method one follows specifically the two-step binding of ATP (scheme 1) with the kinetics: The binding of e-ATP to Sl was monitored in a fluorescence stopped-flow apparatus as in [5]. The excitation and emission wavelengths were 290 and 340 nm, respectively.…”