Adenosine deaminase residual activity

As noted by Kati and Wolfenden, this remarkable affinity appears to suggest that the 6-hydroxyl group, which has very limited freedom of movement, is likely to be in almost ideal alignment with the active site, and that at least one charged active site residue is also likely to be involved in its hydrogen bonding interaction. This conjecture has since been verified by the determination of the crystal structure of the inhibitory complex between adenosine deaminase and 6-hydroxy-1,6-dihydropurine ribonucleoside, which showed that the 6-hydroxyl group interacts with a zinc atom, with a protonated histidyl residue, and with an aspartic acid residue at the enzyme s active site. 

One of these compounds, 9-(p-bromoacetamidobenzyl) adenine, has been used for labeling the amino acid residues of the active site. In fact the functional groups of adenosine deaminase are particularly unreac-tive. The sulfhydryl groups present in the catalytic site react with p-mercuribenzoate and phenylmercuriacetate, but not with haloacetates, haloacetamides, iV-ethylmaleimide, or 5,5 -dithiobis-(2-nitrobenzoic acid). The enzyme is inactivated by l-fluoro-2,4-dinitrobenzene and by haloacetates at pH values over 7.5 and 9.0, respectively more than one lysine residue is modified under these conditions. ... 

The hydrophobic portion of the reagent, but not adenine, appears to be essential for the alkylation of the adenosine deaminase active site since benzylbromoacetate irreversibly inactivates the enzyme whereas the site is inaccessible to haloacetate and its amide. Benzylbromoacetate and 9-(p-bromoacetamidobenzyl) adenine are only 4 and 2.5 more reactive, respectively, than bromoacetate toward 4-(p-nitrobenzyl)pyridine. The oxidation of ribose of purine riboside with periodic acid in order to produce carbonyl groups that may react with lysine residue destroys the inhibitory effectiveness of the nucleoside. ... 

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