Studies on the specificity and mechanism of action of adenosine deaminase

Abstract

The hydrolysis of various groups at the 6-position of derivatives of purine ribonucleoside, including adenosine, 2-amino- and N 6-methyladenosine, 6-chloro-, 6-methoxy-, 6-hydroxylamino-, 2-amino-6-chloro-, and 2-amino-6-methoxypuriue ribonucleoside, by adenosine deamiuase preparations from calf intestinal mucosa has been investigated, and K m and V m values have been determined. Evidence was produced that a single enzyme was involved in the hydrolysis of all these substrates. Thus, purine ribonucleoside inhibited the hydrolysis of each compound in a competitive manner, and K I values for this inhibitor in the presence of various substrates were in the narrow range of 0.75–1.16 × 10 −5 m. Moreover, partial heat denaturation of the enzyme decreased the activity toward each substrate to the same extent. Examination of the effect: of pH on k m suggested dissociating groups on the free enzyme with p K values of 5.3–6.1 and 9.5–10.2, and a dissociating group involving the enzyme substrate complex with a p K of 5.7–6.3. The enzyme was inactivated by p-hydroxymercuribenzoate. The involvement of an SH group at the active center was suggested since this inactivation was antagonized by the competitive inhibitor purine ribonucleoside. The great variation in V m values observed for the various substrates indicated that the rate limiting step was not the final hydrolysis of a common intermediate.