The hydrolytic activity of calf intestinal adenosine deaminase is redu
ced sharply, but reversibly, in the presence of added methanol, ethano
l, acetonitrile, or dioxane. This decrease in k(cat)/K(m) appears to b
e related to diminished water content in the presence of each of these
cosolvents. No agreement between cosolvents is observed if enzyme act
ivity is plotted as a function of viscosity or dielectric constant; no
r do these cosolvents act as conventional reversible inhibitors. The K
(m) value of adenosine and the K(i) values of a substrate analogue (6-
dimethylaminopurine ribonucleoside) and a powerful competitive inhibit
or (6-hydroxy-1,6-dihydropurine ribonucleoside) increase with decreasi
ng solvent water content, but k(cat) is unaffected. Values of 1/K(m) a
nd 1/K(i) increase with roughly the 9th power of the concentration of
water and show no sign of approaching a maximum value as the concentra
tion of water approaches 55 M. These results are consistent with an eq
uilibrium between an abundant, inactive, relatively dehydrated form of
the enzyme and a rare, relatively hydrated form of the enzyme. Only t
he hydrated form of the enzyme, containing at least nine more water mo
lecules than the dehydrated form, appears to be capable of binding sub
strates or competitive inhibitors. Possible physiological consequences
of this behavior, in a tissue in which water is transported in large
quantities, are considered.