Phosphorylation Strongly Affects the Inhibition of Human Carbonic Anhydrase I CO2 Hydration Activity

Human carbonic anhydrases (hCAs) have essential roles in respiration, acid-base balance, and fluid secretion, with implications in diseases such as glaucoma, epilepsy, obesity, and cancer. Of the fifteen known hCAs, human CA I (hCA I) is particularly abundant in erythrocytes, playing a critical role in CO2 transport. Despite extensive research on hCA I, the impact of post-translational modifications (PTMs), particularly phosphorylation, on its catalytic activity and inhibitor binding remains poorly understood. Although multiple phosphorylation sites have been identified in hCA I in vivo through high-throughput proteomics studies including at the highly conserved Ser51 residue, the functional consequences of these modifications are not well characterized. We investigated the effects of a phosphomimic mutation at Ser51 on hCA I, examining its catalytic efficiency and susceptibility to inhibition by sulfonamides and anions. Using a recombinant expression system and a stopped-flow kinetic assay, we characterized the CO2 hydration activity and inhibition profiles of S51E hCA I compared to the wild type enzyme. Our results demonstrate that the S51E mutation increases the catalytic turnover rate (kcat) from 2.0 × 105 s-1 to 2.6 × 105 s-1 but significantly decreases substrate affinity, raising the Michaelis constant (KM) from 4.0 mM to 13.9 mM, reducing overall catalytic efficiency by over 50%. Inhibition studies with a panel of 41 sulfonamides revealed that the S51E mutation dramatically alters inhibitor sensitivity, particularly for the most effective inhibitors. For example, 15 of the 16 most effective sulfonamide inhibitors for hCA I (with KIs <350 nM) were an average of over 35-fold less effective in inhibiting S51E hCA I than the wild type. For example, the KI of the anticonvulsant zonisamide increased from 31 nM for the wild type hCA I to 4 µM. The inhibition profile with a panel of 37 small anions further indicated that the S51E mutant exhibited significantly reduced susceptibility to inhibition by 24 out of 37 tested anions, with some KI values increasing by up to 11,000-fold for inhibitors like hydrogen sulfide. This study underscores the significant impact that phosphorylation may have on hCA I function and inhibition. By characterizing the effects of phosphorylation on the CO2 hydration activity and inhibitor sensitivity of hCA I, these findings represent early steps in developing more selective proteoform-specific inhibitors, which could lead to more effective treatments for diseases involving carbonic anhydrases.