Mechanism of the actomyosin adenosine triphosphatase. Evidence that adenosine 5'-triphosphate hydrolysis can occur without dissociation of the actomyosin complex

Abstract

We have investigated the steps in the actomyosin ATPase cycle that determine the maximum ATPase rate (Vmax) and the binding between myosin subfragment one (S-1) and actin which occurs when the ATPase activity is close to Vmax. We find that the forward rate constant of the initial ATP hydrolysis (initial Pi burst) is about 5 times faster than the maximum turnover rate of the actin S-1 ATPase. Thus, another step in the cycle must be considerably slower than the forward rate of the initial Pi burst. If this slower step occurs only when S-1 is complexed with actin, as originally predicted by the Lymn-Taylor model, the ATPase activity and the fraction of S-1 bound to actin in the steady state should increase almost in parallel as the actin concentration is increased. As measured by turbidity determined in the stopped-flow apparatus, the fraction of S-1 bound to actin, like the ATPase activity, shows a hyperbolic dependence on actin concentration, approaching 100% asymptotically. However, the actin concentration required so that 50% of the S-1 is bound to actin is about 4 times greater than the actin concentration required for half-maximal ATPase activity. Thus, as previously found at 0 degrees C, at 15 degrees C much of the S-1 is dissociated from actin when the ATPase is close to Vmax, showing that a slow first-order transition which follows the initial Pi burst (the transition from the refractory to the nonrefractory state) must be the slowest step in the ATPase cycle. Stopped-flow studies also reveal that the steady-state turbidity level is reached almost instantaneously after the S-1, actin, and ATP are mixed, regardless of the order of mixing. Thus, the binding between S-1 and actin which is observed in the steady state is due to a rapid equilibrium between S-1--ATP and acto--S-1--ATP which is shifted toward acto-S-1--ATP at high actin concentration. Furthermore, both S-1--ATP and S-1--ADP.Pi (the state occurring immediately after the initial Pi burst) appear to have the same binding constant to actin. Thus, at high actin concentration both S-1--ATP and S-1--ADP.Pi are in rapid equilibrium with their respective actin complexes. Although at very high actin concentration almost complete binding of S-1--ATP and S-1--ADP.Pi to actin occurs, there is no inhibition of the ATPase activity at high actin concentration. This strongly suggests that both the initial Pi burst and the slow rate-limiting transition which follows (the transition from the refractory to the nonrefractory state) occur at about the same rates whether the S-1 is bound to or dissociated from actin. We, therefore, conclude that S-1 does not have to dissociate from actin each time an ATP molecule is hydrolyzed.

Effect of cross-bridge kinetics on apparent Ca2+ sensitivity.

“…The fraction of myosin products (X in Eq. 3) in reversible association with actin also increases (Stein et al, 1979) . Because k2 includes the refractory state transition, which is believed to be rate limiting in isolated protein systems (Eisenberg and Greene, 1980), and because the rate of hydrolysis increases in myofibrils with a decrease in ionic strength (over the range 0.1-0.2; Goodno et al, 1978), we argue that reduction in ionic strength decreases the lifetime of the refractory state, i.e., increases k2 .…”

“…A is regulated actin in the thin myofilaments, X is any one of a number of myosin product complexes, 1/k4 is the mean lifetime of the attached state, and 1/k2 is the mean time myosin is detached (weakly attached; Stein et al, 1979) from actin. k_1 (and we assume ka = k_1) is the dissociation rate constant for the Ca-TnC complex .…”

Fluorescence energy transfer between the myosin subfragment‐1 isoenzymes and F‐actin in the absence and presence of nucleotides

“…At the outset of these experiments it was anticipated from the literature that the acto-S1-ADP complex would be indistinguishable from the 'rigor' acto-S1 complex and serve as a control for the experiments with AdoPP[NH]P. This was founded on the observations that many models of the contractile cycle [58,75] predict that actomyosin-ADP complexes are indeed rigor-like. Also X-ray diffraction patterns of whole muscles treated with ADP showed relatively little change from the rigor conformation [76].…”

Isolation and partial renaturation of proteolytic fragments of the myosin head.

“…The affinity of actin for the predominant S-1-nucleotide species was assumed to be ca. 1/5000th of 2 x 108 M-1 (22). Taking Vnmax by NaDodSO4/PAGE.…”