Abstract Hen duodenal adenosine deaminase has been purified from an acetone powder by the following techniques: heating to 58°, fractionation with ammonium sulfate, and column chromatography on epichlorohydrin triethanolamine cellulose and DEAE-Sephadex. The purified material was chromatographically homogeneous on DEAE-Sephadex; electrophoresis of the purified and concentrated enzyme yielded one large enzymatically active band (85%) and another band (15%) which was inactive. The molecular weight of the enzyme was estimated to be 31,000 by use of a calibrated Sephadex G-100 column. The isoelectric point was determined to be 5.0 ± 0.2 pH units by electrophoresis on cellulose acetate strips. The absorption spectrum of the enzyme does not give evidence for a tightly bound prosthetic group and dialysis against ethylenediaminetetraacetate does not lower the enzymatic activity. A readily accessible sulfhydryl group has been titrated with p-mercuribenzoate. Treatment of the native enzyme with 5 µm p-mercuribenzoate produces a stable mercuriated derivative that has an activity 2.5 fold greater than that of the native enzyme. This activation can be reversed by dialysis against dithiothreitol. Higher concentrations of the mercurial give a transient activation and then an irreversible inactivation. Treatment with N-ethylmaleimide (8 x 10-3 m) yields a 1.5-fold activation. The enzyme treated with N-ethylmaleimide is only inactivated by p-mercuribenzoate. These results indicate that there is one very reactive —SH group on the enzyme, and its substitution by either p-mercuribenzoate or N-ethylmaleimide leads to activation. Other groups are inaccessible to N-ethylmaleimide but can react with p-mercuribenzoate to give inactivation. Iodoacetamide does not appear to react with any group on the enzyme. Seven other mercurials were found to have similar activating action, but not all produced stable activated products. There does not seem to be any correlation between the charge on the ligand or its size and the amount of activation. The native and mercuriated enzymes deaminate adenosine, 2'-deoxyadenosine, 3'-deoxyadenosine, 2,6-diaminopurine riboside, and dechlorinate 6-chloropurine riboside. Relative substrate specificity ratios and Michaelis constants were determined for the above substrates. In contrast to most adenosine deaminases, inosine, deoxyinosine, and guanosine give product inhibition with both forms of the enzyme. In addition, purine riboside and 6-mercaptopurine riboside were found to be competitive inhibitors of adenosine, whereas N6-methyl- and N6-dimethyladenosine were not competitive inhibitors. None of the inhibitors were active as substrates. Ammonium sulfate at pH 9 was not inhibitory. All attempts to run the reaction in the reverse direction were unsuccessful. Energies of activation were calculated from Arrhenius plots for adenosine, 2,6-diaminopurine riboside, and 6-chloropurine riboside. Both the slopes and intercepts of Lineweaver-Burk plots were used. The mercuriated enzyme gave considerably lower values than the native enzyme. The energies of activation and the molecular activities were used to determine the ΔF‡, ΔH‡, and ΔS‡ for the slope and intercept terms for both enzymes when adenosine was the substrate. Whereas there is a relatively small change in the ΔF‡ terms upon mercuriation, both the ΔH‡ and ΔS‡ terms decreased strikingly.
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Aug 13, 2010
Thomas P West 
Open Access