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Diethylaminoethyl-cellulose chromatography was used to separate the two isoenzymes of adenosine deaminase (EC3.5.4.4), adenosine deaminase1 (ADA1) and adenosine deaminase2 (ADA2), in human plasma. One hundred and fifteen purine base, nucleoside, and nucleotide analogs were tested as inhibitors of this partially purified preparation of ADA2. Coformycin and 2'-deoxycoformycin were by far the most potent inhibitors of this isoenzyme (apparent Ki values 20 and 19 nM, respectively). ADA2 was also inhibited by nebularine (apparent Ki 1.5 mM) but was resistant to the potent ADA1 inhibitor (+)-erythro-9(2-S-hydroxy-3-R-nonyl)adenine. alpha-D-Adenosine also inhibited ADA2, as did several halogenated purine and adenine base analogs. Structural requirements for the binding of purine analogs to ADA2 are presented which provide a general basis for the design of specific inhibitors of ADA2. Such inhibitors may be useful in studies designed to provide an understanding of the physiological role of ADA2 both in the normal state and in diseases such as human immunodeficiency virus-1 infection where levels in plasma are increased markedly.

1. The scallop enzyme was separated by DE52 ion-exchange chromatography into two forms with the same mol. wt of 38,000 and similar characteristics. 2. The enzyme was inactivated in the absence of dithiothreitol and complete reactivation was achieved by adding the agent within a critical storage period. 3. The apparent values of pKm and Vmax sensitively increased as ionic strength was raised to 250 mM and phosphate and sodium ions elevated the former value with a further increase of the ionic strength. 4. The apparent activation energies for the alpha (Vmax/Km) and beta (Vmax) parameters of both the forms were approximately 5 and 8 kcal/mol, respectively. 5. The enzyme deaminated 2'-, 3'-deoxyadenosine and 2',3'-isopropylidene adenosine but did not deaminate 5'-deoxyadenosine, alpha-adenosine and adenine nucleotides. 6. The affinity for inosine was much lowered with a high Ki value. Adenine and purine riboside inhibited the enzyme completely, and coformycin was a tight, slow binding inhibitor.

The small-size adenosine deaminase (Mr = 35,000 and 43,000) in calf intestinal mucosa, frog liver and scallop adductor muscle and the large-size deaminase (Mr = 100,000) in frog liver probably formed by adding a conversion protein to the small enzyme, were tightly bound to the purine riboside affinity column. Binding of the other large-size enzymes (Mr = 140,000) in the midgut gland of scallop and mussel to the column was not successful. It seems that the binding difference does not depend on a change in affinity between active site and ligand but rather on the stereospecific positioning of active site in the enzyme molecules.

1. The purified mussel enzyme deaminated several adenosine analogs with different Km and relative Vmax values. Affinity for adenine was similar to that for adenosine but the deamination rate was extremely slow. 2. Purine riboside was competitive, coformycin was a tight, slow binding inhibitor, and inhibition by both these compounds was pH-dependent. 3. Inosine, hypoxanthine, guanosine and 6-mercapto-purine riboside were slightly inhibitory. 4. The results suggested that initial binding of the substrate was guided by the adenine moiety followed by a stereospecific steering due to a ribose-dependent distortion in the complex to facilitate nucleophilic attack at C-6.

1. The adenosine deaminase has an approximate molecular weight of 130,000-140,000 and the composition of two polypeptide units (mol. wt about 68,000) is suggested, by means of SDS disc electrophoresis. 2. Both the alpha (Vm/Km) and beta (Vm) parameters were varied with pH and temperature. RSS (relative substrate specificity) adenosine and deoxyadenosine values for alpha and beta were 1.2 and 1.1, respectively. 3. Adenine, 2'-, 3', 5'-AMP, 5'-deoxyAMP, ADP and ATP were not deaminated by the enzyme. 4. Inhibition by Mg2+ was found in reaction with adenosine at pH 8 but not with deoxyadenosine at the same pH. Mn2+, which did not affect the reaction rate at pH 4 and 5, showed competitive inhibitory effects at pH 6, 7 and 8.

Adenosine deaminase (ADA) was partially purified from several sources using affinity chromatography. These enzymes have the capacity to catalyze the deamination of 2,6-diamino-9-(2-hydroxyethoxymethyl)purine (A134U) to form the antiviral agent acyclovir [9-(2-hydroxyethoxymethyl)guanine]. Their relative substrate efficiencies (Vmax/Km) with A134U (standardized to adenosine = 100) were: dog ADA, 0.092; human ADA, 0.015-0.029; rat ADA, 0.025; calf ADA, 0.016; and Escherichia coli ADA, 0.0003. In addition to having the lowest efficiency with A134U, the bacterial ADA was also distinguished by its lack of binding of the mammalian ADA inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine and by its weak binding to the 9-(p-aminobenzyl)adenine-agarose affinity column. Four minor metabolites of A134U and acyclovir have been reported to be produced in the rat. These compounds are oxidized on either the C-8 position of the ring or the terminal carbon of the side chain. Neither acyclovir nor any of these metabolites produced significant inhibition of calf intestine ADA. The oxidized metabolites containing an N-6 amino group were extremely slow substrates of this enzyme.