title: 2',3'-cAMP hydrolysis by metal-dependent phosphodiesterases containing DHH, EAL, and HD domains is non-specific: Implications for PDE screening.
abstract: The recent report of 2',3'-cAMP isolated from rat kidney is the first proof of its biological existence, which revived interest in this mysterious molecule. 2',3'-cAMP serves as an extracellular adenosine source, but how it is degraded remains unclear. Here, we report that 2',3'-cAMP can be hydrolyzed by six phosphodiesterases containing three different families of hydrolytic domains, generating invariably 3'-AMP but not 2'-AMP. The catalytic efficiency (k(cat)/K(m)) of each enzyme against 2',3'-cAMP correlates with that against the widely used non-specific substrate bis(p-nitrophenyl)phosphate (bis-pNPP), indicating that 2',3'-cAMP is a previously unknown non-specific substrate for PDEs. Furthermore, we show that the exclusive formation of 3'-AMP is due to the P-O2' bond having lower activation energy and is not the result of steric exclusion at enzyme active site. Our analysis provides mechanistic basis to dissect protein function when 2',3'-cAMP hydrolysis is observed.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore. RAOF0001@ntu.edu.sg
authors.forename: Feng
authors.initials: F
authors.lastname: Rao
authors.forename: Yaning
authors.initials: Y
authors.lastname: Qi
authors.forename: Elavazhagan
authors.initials: E
authors.lastname: Murugan
authors.forename: Swathi
authors.initials: S
authors.lastname: Pasunooti
authors.forename: Qiang
authors.initials: Q
authors.lastname: Ji
mesh_terms: D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D006801:Humans; D006868:Hydrolysis; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D013379:Substrate Specificity
chemical_list: D000227:Adenine Nucleotides; D008667:Metalloproteins; C010565:2',3'-cyclic AMP; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases