Determinants for the cAMP-binding site at the S-adenosylhomocysteine-hydrolase.
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.
Kloor
Doris
D
Department of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Tübingen, Wilhelmstrasse 56, Tübingen, Germany. doris.kloor@uni-tuebingen.de
Hermes
Marina
M
Kirschler
Julia
J
Müller
Margret
M
Hagen
Norbert
N
Kalbacher
Hubert
H
Stevanovic
Stefan
S
Osswald
Hartmut
H
D000241:Adenosine / Q000378:metabolism; D043383:Adenosylhomocysteinase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D000242:Cyclic AMP / Q000008:administration & dosage / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D019920:Photoaffinity Labels; D011485:Protein Binding; D012435:S-Adenosylhomocysteine / Q000378:metabolism*
D019920:Photoaffinity Labels; D012435:S-Adenosylhomocysteine; D000242:Cyclic AMP; D043383:Adenosylhomocysteinase; D000241:Adenosine