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

Doris Kloor1, Marina Hermes, Julia Kirschler

  • 1Department 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
|June 24, 2009
PubMed

Insights

Cyclic adenosine monophosphate (cAMP) enhances S-adenosylhomocysteine-hydrolase (AdoHcy-hydrolase) activity by binding to adenosine's site. This binding is independent of the enzyme's redox state, suggesting a novel regulatory mechanism.

Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • S-Adenosylhomocysteine-hydrolase (AdoHcy-hydrolase) is crucial for homocysteine metabolism.
  • Adenosine (Ado) competes with cyclic adenosine monophosphate (cAMP) for the enzyme's binding site.
  • Understanding cAMP's role is key to elucidating AdoHcy-hydrolase regulation.

Purpose of the Study:

  • To investigate the effect of cAMP on AdoHcy-hydrolase activity and binding characteristics.
  • To determine if cAMP acts as an allosteric effector or competes with substrates.
  • To identify the cAMP binding site on AdoHcy-hydrolase.

Main Methods:

  • Enzyme kinetics assays with purified bovine kidney AdoHcy-hydrolase (native, NAD(+), NADH forms).
  • Photoaffinity labeling using 8-azido-[2-(3)H]-cAMP.
  • Peptide isolation and sequencing to identify the cAMP binding site.

Main Results:

  • cAMP (10 µmol/l) enhanced native AdoHcy-hydrolase activity by 35%; no stimulation observed for the NAD(+) form.
  • cAMP binding did not inhibit enzymatic activity and did not prevent Ado-induced inhibition.
  • Photoaffinity labeling identified a single high-affinity binding site for cAMP, located within the NAD-binding domain (Trp310-Val325), independent of the enzyme's redox state.

Conclusions:

  • The cAMP binding site on AdoHcy-hydrolase is distinct from the catalytic site and identical to the high-affinity Ado binding site.
  • cAMP does not function as an allosteric effector for AdoHcy-hydrolase.
  • cAMP binding is independent of the enzyme's NAD(+)/NADH ratio, suggesting a novel regulatory pathway.

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