2a super(2),3a super(2)-cAMP hydrolysis by metal-dependent phosphodiesterases containing DHH, EAL, and HD domains is non-specific: Implications for PDE screening

• Published in: Biochemical and biophysical research communications Vol. 398; no. 3; pp. 500 - 505
• Main Authors: Rao, Feng, Qi, Yaning, Murugan, Elavazhagan, Pasunooti, Swathi, Ji, Qiang
• Format: Journal Article
• Language: English
• Published: 30-07-2010
• Subjects: Adenosine
• ISSN: 0006-291X
• DOI: 10.1016/j.bbrc.2010.06.107

The recent report of 2a super(2),3a super(2)-cAMP isolated from rat kidney is the first proof of its biological existence, which revived interest in this mysterious molecule. 2a super(2),3a super(2)-cAMP serves as an extracellular adenosine source, but how it is degraded remains unclear. Here, we report that 2a super(2),3a super(2)-cAMP can be hydrolyzed by six phosphodiesterases containing three different families of hydrolytic domains, generating invariably 3a super(2)-AMP but not 2a super(2)-AMP. The catalytic efficiency (k cat/K m) of each enzyme against 2a super(2),3a super(2)-cAMP correlates with that against the widely used non-specific substrate bis(p-nitrophenyl)phosphate (bis-pNPP), indicating that 2a super(2),3a super(2)-cAMP is a previously unknown non-specific substrate for PDEs. Furthermore, we show that the exclusive formation of 3a super(2)-AMP is due to the P-O2a super(2) 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 2a super(2),3a super(2)-cAMP hydrolysis is observed.