Characterization of the cAMP binding site of purified S-adenosyl-homocysteine hydrolase from bovine kidney

The enzyme S-adenosyl-homocysteine hydrolase (AdoHcyase) which catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine is an adenosine binding protein. In the present study we examined the characteristics of [ 3H]cAMP binding to purified AdoHcyase from bovine kidney in comparison with the high affinity adenosine binding site of AdoHcyase. AdoHcyase exhibits one [ 3H]cAMP binding site with an affinity of K d =23.1±1.1 nM and a B max of 116.6±3.8 pmol/mg protein. Binding of [ 3H]cAMP obeyed a monophasic reaction with a k +1 value of 0.035 min/M. The dissociation of AdoHcyase–[ 3H]cAMP complex exhibited a time- and temperature-dependent character. After a 240 min incubation at 0° only 5–10%, however, at 20° 90% were displaceable. Adenosine and cAMP displace each other with similar affinities of ec 50 57 nM vs. ec 50 65 nM. 2′-Deoxyadenosine, N 6-methyladenosine, and NECA displace 25 nM [ 3H]cAMP and 10 nM [ 3H]adenosine with ec 50 values of 94, 90 and 80 nM, respectively. All other nucleosides studied, adenine, inosine, adenosine-2′,3′-dialdehyde, 2-chloroadenosine, aristeromycin, and adenine nucleotides were only week competitors for [ 3H]cAMP and [ 3H]adenosine. These compounds displace [ 3H]cAMP and [ 3H]adenosine with equal potencies. Our data indicate that the binding site for nanomolar concentrations of cAMP and adenosine at the AdoHcyase appears to be identical. The physiological implications of a cAMP binding site at the AdoHcyase remain to be established.