Abstract
The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'-(3-thiotriphosphate) in the presence of Mg(2+) are incompatible with a simple bimolecular association process. A substrate-induced conformation change with DeltaG(0)<-24kJ.mol(-1) (i.e. DeltaG(0) could be more negative) at pH8 and 21 degrees C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATPright harpoon over left harpoonM.ATPright harpoon over left harpoonM*.ATP, where the association constant for the first step, K(1), is 4.5x10(3)m(-1) at I 0.14m and the rate of isomerization is 400s(-1). In the presence of Mg(2+), ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s(-1), but with DeltaG(0) for that process being -14kJ.mol(-1). The effect of increasing ionic strength is to decrease K(1), the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.
Highlights
The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'43-thiotriphosphate) in the presence of Mg2+ are incompatible with a simple bimolecular association process
By rapid-reaction-kinetics methods four intermediates have been characterized during the catalysis of ATP hydrolysis by heavy meromyosin (Trentham et al, 1972)
An important feature of the ATPase mechanism is that when ATP is mixed with heavy meromyosin, there occurs a transient in the formation of the product-protein complex equal to about 0.8mol/mol of ATPase site
Summary
The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5'43-thiotriphosphate) in the presence of Mg2+ are incompatible with a simple bimolecular association process. In the presence of Mg2+, ADP binds in a similar fashion to ATP, the rate of the conformation change being 400s-1, but with AGO for that process being -14kJmol-'. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants. An important feature of the ATPase mechanism is that when ATP is mixed with heavy meromyosin, there occurs a transient in the formation of the product-protein complex equal to about 0.8mol/mol of ATPase site. 0.1 mM the cleavage of ATP occurs with an apparent second-order rate constant of 1 x 106-2 x 106M-1 * S-1, but at high ATP concentrations the observed cleavage rate becomes independent of ATP concentration and approximately equal to 160s-I
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