Studies on Adenosine Deaminase

The substrate specificity of adenosine deaminase purified from the maternal component of the bovine placenta has been studied in detail, and the effects of different types of inhibitors have been examined. The enzyme catalysed the hydrolytic removal of amino, chloro, hydroxylamino, methoxy and methoxyamino substituents from the 6 position of purine ribonuclesides; 2‐substitution by amino, chloro or fluoro groups reduced the V of adenosine and 6‐chloropurineriboside but 2‐amino substitution increased the V of 6‐methoxypurineriboside. The results indicate that both a bond‐forming component dependent on steric factors, and a bond‐stretching component dependent on the electronegativity of the leaving group are involved in the rate‐determining formation of the transition complex. 2‐Alkylamino‐, 2‐alkylthio‐, and 2‐halogenoadenosines are competitive inhibitors with K i values which conform the importance of the basicity of N 1 of substrates and inhibitors in their binding to the active site of the enzyme. The enzyme was shown to have dissociating groups with p K a values of 9.8, 8.9 and 5.7. Enzyme activity was inhibited by p ‐chloromercuribenzote (PCMB), and titration of the native enzyme with this mercurial showed that there are two sulphydryl groups per mole of enzyme. There was no reaction of the enzyme with the sulphydryl reagent, 5,5′‐dithiobis‐(2‐nitrobenzoic acid). 2‐Mercaptoethanol and glutathione in high concentrations inhibited the enzyme; inhibition by glutathione was irreversible.