Carbonic anhydrases (CAs) are a metalloenzyme family that have important roles in cellular processes including pH homeostasis and have been implicated in multiple pathological conditions. Small molecule inhibitors have been developed to target carbonic anhydrases, but the effects of post-translational modifications (PTMs) on the activity and inhibition profiles of these enzymes remain unclear. Here, we investigate the effects of phosphorylation, the most prevalent carbonic anhydrase PTM, on the activities and drug-binding affinities of human CAI and CAII, two heavily modified active isozymes. Using serine to glutamic acid (S > E) mutations to mimic the effect of phosphorylation, we demonstrate that phosphomimics at a single site can significantly increase or decrease the catalytic efficiencies of CAs, depending on both the position of the modification and the CA isoform. We also show that the S > E mutation at Ser50 of hCAII decreases the binding affinities of hCAII with well-characterized sulphonamide inhibitors including by over 800-fold for acetazolamide. Our findings suggest that CA phosphorylation may serve as a regulatory mechanism for enzymatic activity, and affect the binding affinity and specificity of small, drug and drug-like molecules. This work should motivate future studies examining the PTM-modification forms of CAs and their distributions, which should provide insights into CA physiopathological functions and facilitate the development of 'modform-specific' carbonic anhydrase inhibitors.
Keywords: carbonic anhydrase inhibition; esterase activity; human carbonic anhydrase I; human carbonic anhydrase II; phosphorylation; post-translational modifications; sulphonamides.