Determinants for the cAMP-binding site at the S-adenosylhomocysteine-hydrolase.

Doris Kloor, Marina Hermes, Julia Kirschler, Margret Müller, Norbert Hagen, Hubert Kalbacher, Stefan Stevanovic, Hartmut Osswald

Department of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Tübingen, Wilhelmstrasse 56, Tübingen, Germany. doris.kloor@uni-tuebingen.de

Naunyn-Schmiedeberg's archives of pharmacology 2009 Sep

filter terms:

S-Adenosylhomocysteine-hydrolase (AdoHcy-hydrolase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy). Since Ado competes with cAMP at the high affinity-binding site of the enzyme, we determined the effect of cAMP on enzyme activity and its binding characteristics to purified AdoHcy-hydrolase from bovine kidney in its native, in its fully oxidized (NAD(+)), and in its fully reduced (NADH) form. cAMP (10 micromol/l) enhanced the hydrolytic activity of native AdoHcy-hydrolase by 35%, whereas the activity of the enzyme in its NAD(+) form was not stimulated by cAMP. In contrast to azido-Ado, binding of azido-cAMP did not inhibit the enzymatic activity of AdoHcy-hydrolase. Furthermore, cAMP did not prevent the Ado induced inhibition of the AdoHcy hydrolysis. Saturation binding experiments with the three different forms of AdoHcy-hydrolase, native, NAD(+), and NADH showed only one binding site with high affinity. This binding site was identified after photoaffinity labeling of the enzyme with 8-azido-[2-(3)H]-cAMP. One photolabeled peptide was isolated as Trp(310)-Val(325) from each AdoHcy-hydrolase from native, NAD(+), and NADH. The cAMP-labeled peptide is located in the NAD-binding domain of AdoHcy-hydrolase. In conclusion, our data show that the cAMP-binding site at the AdoHcy-hydrolase is independent of the NAD(+)/NADH ratio of the enzyme and is identical with the high affinity-binding site of Ado. Moreover, cAMP did not interact with the catalytic site of AdoHcy-hydrolase and did not act as an allosteric effector for the AdoHcy-hydrolase.

Citation

Doris Kloor, Marina Hermes, Julia Kirschler, Margret Müller, Norbert Hagen, Hubert Kalbacher, Stefan Stevanovic, Hartmut Osswald. Determinants for the cAMP-binding site at the S-adenosylhomocysteine-hydrolase. Naunyn-Schmiedeberg's archives of pharmacology. 2009 Sep;380(3):215-22