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

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.