Inactivation of Myosin by 2,4-dinitrophenol and Protection by Adenosine Triphosphate and Other Phosphate Compounds.

It has become apparent that adenosine triphosphate binds to the active site of myosin in a way which is not optimal for hydrolytic cleavage of the terminal phosphate bond. Depending on conditions, a wide variety of reagents, including 2,4-dinitrophenol, can improve the catalysis of this reaction by altering the interaction between enzyme and substrate. Concerning the way in which these modifiers affect the myosin-ATP complex, there are two general points of view which are not exclusive of each other. First, it is often assumed that the modifier directly alters substrate binding by interacting at the active site in place of some group on the substrate. If this is true, then the chemical nature of the modifying reagent tells us something about the groups at the active site. For example, reasoning along these lines, there appear to be at least two -SH groups at the active site of myosin. This follows from observations that a number of SH-binding reagents (2-5) activate myosin by binding up to half its SH groups; these same compounds begin to inhibit when they mask the remaining half. There is, then, by this view at least one rateretarding and one rate-promoting -SH at the active center; blocking the former activates whereas blocking the latter inhibits. Similar effects of other reagents have been used to implicate an amino group of lysine at the active site (to explain inhibition by 2,4,6-trinitrobenzenesulfonate) (6) and 2 bound metal ions (inferred from the effects of reagents like inorganic pyrophosphate and ethylenediaminetetraacetate) (7). Moreover, the complex effects of 2,4-dinitrophenol on myosin adenosine triphosphatase might involve still other unknown groups at the hydrolytic site 03). A different point of view arises when we consider the similar activating and inhibiting effects on myosin of reagents like urea, guanidine, ethylene glycol (9), aliphatic alcohols (lo), and dioxane (9, ll), some of which have been shown to alter the physical properties of the enzyme at concentrations which activate. According to this line of reasoning, the modifiers operate to change the conformation of the protein, thus indirectly altering the orientation of the enzyme-substrate complex. The effect of modifiers on the temperature dependence of ATP hydrolysis by myosin has been interpreted in similar terms (12). I f this view is true, the chemical nature of the modifier does not tell us explicitly