Rate-limiting step in the actomyosin adenosine triphosphatase cycle: experiments with myosin subfragment 1 crosslinked to actin

Abstract

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)

Biochemical kinetics of porcine cardiac subfragment-1. II. Pre-steady-state studies of the initial phosphate burst.

“…The reduced magnitudes in Figure 5 are corrected magnitudes in that they have been divided by the irreversible binding magnitude (0.7-0.8 mol Pj/mol S-l). 1 The magnitudes reported above are in close agreement with those originally reported by Stein et al 5 for skeletal S-l and also with those more recently reported by Rosenfeld and Taylor 12 for skeletal S-l (see "Discussion").…”

“…4 ' 5 This lack of significant inhibition of the ATPase activity of cardiac actoS-1 at saturating actin con-centrations was reported previously for skeletal S-l, and detailed kinetic modeling revealed that a "nondissociating pathway" for ATP hydrolysis was required to account for the data. 5 It was then concluded that the four-state model was the minimal model that could account for the data (see Figure I). 1 Because the steady-state kinetics of cardiac S-l are almost identical to rabbit skeletal S-l, except for a significantly different V^, it becomes interesting to pursue the kinetic mechanism of this difference.…”

“…The fluorescence increase has been used in the past as a measure of the burst magnitude. 5 The implicit assumption is that states AM**DP, and M**DP, have the same tryptophan fluorescence enhancement associated with them (also true for AM*T and M*T). Evidence that this may be the case does exist.…”

“…4 ' 5 This lack of significant inhibition of the ATPase activity of cardiac actoS-1 at saturating actin con- centrations was reported previously for skeletal S-l, and detailed kinetic modeling revealed that a "nondissociating pathway" for ATP hydrolysis was required to account for the data. 5 It was then concluded that the four-state model was the minimal model that could account for the data (see Figure I).…”

Is a four‐state model sufficient to describe actomyosin ATPase?

“…[12]). Thus, it is proposed that in the four-state model, the kinetics of the hydrolysis and release of Pi steps are simifar and, therefore, that the Pr burst is small and in the steady-state, actomyosinsubstrate complexes predominate.…”