288 BIOPHYSICAL STUDIES ON NERVE AND MUSCLE 



concentrations, 



v k 2 [E] n [S] a 

 [S] + K m 



(see Chapter 8 for symbols), is well obeyed. Measurement of rate as a func- 

 tion of temperature and substrate concentration permits evaluation of AH t , 

 AF l and AS 1 , the thermodynamic quantities associated with formation of 

 the activated state. Since AS* is usually (for various conditions) found to be 

 positive, it is inferred that a change in configuration of the enzyme (and/or 

 the release of adsorbed water molecules) occurs during the binding step in 

 which an ATP molecule sits down on the myosin molecule. This step is then 

 followed by the splitting reaction proper. In the terminology discussed in 

 Chapter 8 and illustrated in Figure 8-5: 



E + S y^ ES l — ^ product 



in which process 1 is adsorption and shortening; and process 2 is the hy- 

 drolysis step. 



When experimental conditions are such that the kinetic results are amen- 

 able to analysis without ambiguity of mechanism, analysis shows that the 

 binding of the (enzyme) myosin molecule to the (substrate) ATP molecule 

 occurs spontaneously with release of 6.6 kcal/mole. That is, 



A/7 bind,n g = -6.6 kcal/mole 

 and 



A// binding = -8.0 kcal/mole 



Thus the free energy released in the binding process is a sizable fraction of 

 that for the whole process (— 10.5). This indicates that the structural change 

 (shortening) of the myosin molecule may occur at the time of binding of ATP, 

 before ATP is split by hydrolysis. The inference is, then, that the resting 

 muscle is very much like a stretched molecular spring, ready to contract 

 when released from the forces which hold it extended. Indeed X-ray diffrac- 

 tion patterns suggest that the famous alpha helix, discussed in Chapter 6, is 

 the basic structure in myosin, as well as in so many other proteins. 



Studies of effects of pressure and of dielectric constant on the rate have 

 given values of the entropy of complex formation (i.e., of enzyme-substrate 

 binding) to be A.S' bindj ~ 48 cal/deg. mole, with half of this value purely 

 electrostatic, due to the charged groups on ATP and myosin. 



Under certain experimental conditions the rate of desorption of the hy- 

 drolytic fragments is slow, causing inhibition by the products. Activators 

 and inhibitors can complicate the picture much further. However, enough 

 has been shown to illustrate the fact that the kinetic methods, although very 



