{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T15:13:20Z","timestamp":1776352400179,"version":"3.51.2"},"reference-count":53,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2023,5,25]],"date-time":"2023-05-25T00:00:00Z","timestamp":1684972800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000923","name":"Australian Research Council","doi-asserted-by":"publisher","award":["DP190103298"],"award-info":[{"award-number":["DP190103298"]}],"id":[{"id":"10.13039\/501100000923","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000923","name":"Australian Research Council","doi-asserted-by":"publisher","award":["FT200100798"],"award-info":[{"award-number":["FT200100798"]}],"id":[{"id":"10.13039\/501100000923","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 &gt; 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 &gt; 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 \u2018modform-specific\u2019 carbonic anhydrase inhibitors.<\/jats:p>","DOI":"10.3390\/ijms24119275","type":"journal-article","created":{"date-parts":[[2023,5,26]],"date-time":"2023-05-26T02:05:44Z","timestamp":1685066744000},"page":"9275","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Effects of Phosphorylation on the Activity, Inhibition and Stability of Carbonic Anhydrases"],"prefix":"10.3390","volume":"24","author":[{"given":"Xiaojing","family":"Huang","sequence":"first","affiliation":[{"name":"School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel","family":"Winter","sequence":"additional","affiliation":[{"name":"School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dominic J.","family":"Glover","sequence":"additional","affiliation":[{"name":"School of Biotechnology & Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4262-0323","authenticated-orcid":false,"given":"Claudiu T.","family":"Supuran","sequence":"additional","affiliation":[{"name":"Neurofarba Department, Sezione di Scienze Farmaceutiche, Universita degli Studi di Firenze, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6622-8193","authenticated-orcid":false,"given":"William A.","family":"Donald","sequence":"additional","affiliation":[{"name":"School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,5,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Mboge, M.Y., Mahon, B.P., McKenna, R., and Frost, S.C. (2018). Carbonic Anhydrases: Role in pH Control and Cancer. Metabolites, 8.","DOI":"10.3390\/metabo8010019"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1570","DOI":"10.1016\/j.bmc.2012.04.044","article-title":"Structure, function and applications of carbonic anhydrase isozymes","volume":"21","author":"Hassan","year":"2013","journal-title":"Bioorg. Med. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4963","DOI":"10.1113\/jphysiol.2013.251181","article-title":"Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1\/SLC4A1","volume":"591","author":"Papadopoulos","year":"2013","journal-title":"J. Physiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2414","DOI":"10.1021\/bi051132d","article-title":"A Novel Carbonic Anhydrase II Binding Site Regulates NHE1 Activity","volume":"45","author":"Li","year":"2006","journal-title":"Biochemistry"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1146\/annurev.ph.58.030196.002515","article-title":"Multiple Roles of Carbonic Anhydrase in Cellular Transport and Metabolism","volume":"58","author":"Henry","year":"1996","journal-title":"Annu. Rev. Physiol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Frost, S.C., and McKenna, R. (2014). Carbonic Anhydrase: Mechanism, Regulation, Links to Disease, and Industrial Applications, Springer.","DOI":"10.1007\/978-94-007-7359-2"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Ciccone, L., Cerri, C., Nencetti, S., and Orlandini, E. (2021). Carbonic Anhydrase Inhibitors and Epilepsy: State of the Art and Future Perspectives. Molecules, 26.","DOI":"10.3390\/molecules26216380"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Poggetti, V., Salerno, S., Baglini, E., Barresi, E., Da Settimo, F., and Taliani, S. (2022). Carbonic Anhydrase Activators for Neurodegeneration: An Overview. Molecules, 27.","DOI":"10.3390\/molecules27082544"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1038\/nrd2467","article-title":"Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators","volume":"7","author":"Supuran","year":"2008","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"825","DOI":"10.2174\/156802607780636690","article-title":"Carbonic anhydrases as drug targets\u2014An overview","volume":"7","author":"Supuran","year":"2007","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1450","DOI":"10.1080\/14756366.2020.1781846","article-title":"Human carbonic anhydrases and post-translational modifications: A hidden world possibly affecting protein properties and functions","volume":"35","author":"Supuran","year":"2020","journal-title":"J. Enzyme Inhib. Med. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"D512","DOI":"10.1093\/nar\/gku1267","article-title":"PhosphoSitePlus, 2014: Mutations, PTMs and recalibrations","volume":"43","author":"Hornbeck","year":"2015","journal-title":"Nucleic Acids Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"D451","DOI":"10.1093\/nar\/gky1052","article-title":"qPhos: A database of protein phosphorylation dynamics in humans","volume":"47","author":"Yu","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"23534","DOI":"10.1038\/srep23534","article-title":"dbPAF: An integrative database of protein phosphorylation in animals and fungi","volume":"6","author":"Ullah","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"R84","DOI":"10.1016\/S0960-9822(00)00287-6","article-title":"Ubiquitination: RING for destruction?","volume":"10","author":"Freemont","year":"2000","journal-title":"Curr. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"90739","DOI":"10.1039\/C6RA08283A","article-title":"Non-enzymatic glycation mediated structure\u2013function changes in proteins: Case of serum albumin","volume":"6","author":"Awasthi","year":"2016","journal-title":"RSC Adv."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1016\/j.ijbiomac.2020.01.192","article-title":"Study of glycation process of human carbonic anhydrase II as well as investigation concerning inhibitory influence of 3-beta-hydroxybutyrate on it","volume":"149","author":"Gharib","year":"2020","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2628","DOI":"10.1038\/sj.onc.1203481","article-title":"Serine phosphorylation of STATs","volume":"19","author":"Decker","year":"2000","journal-title":"Oncogene"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"747","DOI":"10.1016\/S0960-9822(95)00151-5","article-title":"Transcriptional control by protein phosphorylation: Signal transmission from the cell surface to the nucleus","volume":"5","author":"Karin","year":"1995","journal-title":"Curr. Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1007\/s00360-015-0942-4","article-title":"Phosphorylation increases the catalytic activity of rainbow trout gill cytosolic carbonic anhydrase","volume":"186","author":"Carrie","year":"2016","journal-title":"J. Comp. Physiol. B"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Blanco-Rivero, A., Shutova, T., Rom\u00e1n, M.J., Villarejo, A., and Martinez, F. (2012). Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0049063"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4421","DOI":"10.1021\/cr200176r","article-title":"Multiple Binding Modes of Inhibitors to Carbonic Anhydrases: How to Design Specific Drugs Targeting 15 Different Isoforms?","volume":"112","author":"Alterio","year":"2012","journal-title":"Chem. Rev."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Duan, G., and Walther, D. (2015). The roles of post-translational modifications in the context of protein interaction networks. PLoS Comput. Biol., 11.","DOI":"10.1371\/journal.pcbi.1004049"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"231","DOI":"10.3109\/14756366.2012.700640","article-title":"Carbonic anhydrase III: A neglected isozyme is stepping into the limelight","volume":"28","author":"Harju","year":"2013","journal-title":"J. Enzyme Inhib. Med. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1016\/0167-4838(92)90412-7","article-title":"Proton transfer roles of lysine 64 and glutamic acid 64 replacing histidine 64 in the active site of human carbonic anhydrase II","volume":"1122","author":"Engstrand","year":"1992","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2267","DOI":"10.1016\/j.bmcl.2008.03.012","article-title":"Investigations of the esterase, phosphatase, and sulfatase activities of the cytosolic mammalian carbonic anhydrase isoforms I, II, and XIII with 4-nitrophenyl esters as substrates","volume":"18","author":"Innocenti","year":"2008","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"955","DOI":"10.3109\/14756366.2014.1001754","article-title":"Esterase activity of carbonic anhydrases serves as surrogate for selecting antibodies blocking hydratase activity","volume":"30","author":"Uda","year":"2015","journal-title":"J. Enzyme Inhib. Med. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"782","DOI":"10.1177\/1087057106289403","article-title":"Inhibition profiling of human carbonic anhydrase II by high-throughput screening of structurally diverse, biologically active compounds","volume":"11","author":"Iyer","year":"2006","journal-title":"J. Biomol. Screen."},{"key":"ref_29","first-page":"580","article-title":"Some drugs inhibit in vitro hydratase and esterase activities of human carbonic anhydrase-I and II","volume":"59","author":"Ekinci","year":"2007","journal-title":"Pharmacol. Rep. PR"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1016\/j.chembiol.2015.01.001","article-title":"Phosphomimics Destabilize Hsp27 Oligomeric Assemblies and Enhance Chaperone Activity","volume":"22","author":"Jovcevski","year":"2015","journal-title":"Chem. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"652a","DOI":"10.1016\/j.bpj.2012.11.3602","article-title":"Huntington-Associated Phosphorylation of Kinesin-1 Enhances Autoinhibition in a Phosphomimic","volume":"104","author":"Hackney","year":"2013","journal-title":"Biophys. J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"821","DOI":"10.1002\/jms.1581","article-title":"The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry","volume":"44","author":"Gropengiesser","year":"2009","journal-title":"J. Mass Spectrom."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Duong-Ly, K.C., and Peterson, J.R. (2013). The Human Kinome and Kinase Inhibition as a therapeutic strategy. Curr. Protoc. Pharmacol., 2.","DOI":"10.1002\/0471141755.ph0209s60"},{"key":"ref_34","unstructured":"Shenolikar, S. (2012). Progress in Molecular Biology and Translational Science, Academic Press."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/S0962-8924(97)01172-0","article-title":"Integrin signalling and tyrosine phosphorylation: Just the FAKs?","volume":"8","author":"Schlaepfer","year":"1998","journal-title":"Trends Cell Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1192","DOI":"10.1016\/0022-2836(92)90531-N","article-title":"Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes","volume":"227","author":"Carlsson","year":"1992","journal-title":"J. Mol. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Sitbon, E., and Pietrokovski, S. (2007). Occurrence of protein structure elements in conserved sequence regions. BMC Struct. Biol., 7.","DOI":"10.1186\/1472-6807-7-3"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1038\/nature18003","article-title":"Proteogenomics connects somatic mutations to signalling in breast cancer","volume":"534","author":"Mertins","year":"2016","journal-title":"Nature"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"876","DOI":"10.1038\/ncomms1871","article-title":"Quantitative maps of protein phosphorylation sites across 14 different rat organs and tissues","volume":"3","author":"Lundby","year":"2012","journal-title":"Nat. Commun."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2414","DOI":"10.1021\/pr300825v","article-title":"Identification of missing proteins in the neXtProt database and unregistered phosphopeptides in the PhosphoSitePlus database as part of the Chromosome-centric Human Proteome Project","volume":"12","author":"Shiromizu","year":"2013","journal-title":"J. Proteome Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1351","DOI":"10.1006\/jmbi.1999.3310","article-title":"Sequence and structure-based prediction of eukaryotic protein phosphorylation sites","volume":"294","author":"Blom","year":"1999","journal-title":"J. Mol. Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"52","DOI":"10.4161\/cc.4.1.1353","article-title":"Protein kinase specificity. A strategic collaboration between kinase peptide specificity and substrate recruitment","volume":"Tex 4","author":"Zhu","year":"2005","journal-title":"Cell Cycle Georget."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1006\/jmbi.1994.1491","article-title":"Enzyme-substrate interactions. Structure of human carbonic anhydrase I complexed with bicarbonate","volume":"241","author":"Kumar","year":"1994","journal-title":"J. Mol. Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/gigascience\/giw015","article-title":"Estimating the total number of phosphoproteins and phosphorylation sites in eukaryotic proteomes","volume":"6","author":"Vlastaridis","year":"2017","journal-title":"GigaScience"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1248","DOI":"10.1016\/S1044-0305(98)00103-2","article-title":"Unfolding of proteins monitored by electrospray ionization mass spectrometry: A comparison of positive and negative ion modes","volume":"9","author":"Konermann","year":"1998","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e25068","DOI":"10.4161\/idp.25068","article-title":"Extracting structural information from charge-state distributions of intrinsically disordered proteins by non-denaturing electrospray-ionization mass spectrometry","volume":"1","author":"Testa","year":"2013","journal-title":"Intrinsically Disord. Proteins"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"4245","DOI":"10.1021\/acs.jmedchem.5b01643","article-title":"Kinetic and Structural Insights into the Mechanism of Binding of Sulfonamides to Human Carbonic Anhydrase by Computational and Experimental Studies","volume":"59","author":"Gaspari","year":"2016","journal-title":"J. Med. Chem."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1021\/acscentsci.8b00787","article-title":"Nanoscale Ion Emitters in Native Mass Spectrometry for Measuring Ligand\u2013Protein Binding Affinities","volume":"5","author":"Tran","year":"2019","journal-title":"ACS Cent. Sci."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"4614","DOI":"10.1021\/acs.analchem.0c00163","article-title":"Perfluoroalkyl Substances of Significant Environmental Concern Can Strongly Inhibit Human Carbonic Anhydrase Isozymes","volume":"92","author":"Nocentini","year":"2020","journal-title":"Anal. Chem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"561","DOI":"10.1080\/14756366.2021.1882453","article-title":"Reconsidering anion inhibitors in the general context of drug design studies of modulators of activity of the classical enzyme carbonic anhydrase","volume":"36","author":"Nocentini","year":"2021","journal-title":"J. Enzyme Inhib. Med. Chem."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1038\/nmeth.1318","article-title":"Enzymatic assembly of DNA molecules up to several hundred kilobases","volume":"6","author":"Gibson","year":"2009","journal-title":"Nat. Methods"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"2658","DOI":"10.1038\/srep02658","article-title":"Quantitative full time course analysis of nonlinear enzyme cycling kinetics","volume":"3","author":"Cao","year":"2013","journal-title":"Sci. Rep."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"2241","DOI":"10.1016\/S0968-0896(03)00112-3","article-title":"Hydroxyurea is a carbonic anhydrase inhibitor","volume":"11","author":"Scozzafava","year":"2003","journal-title":"Bioorg. Med. Chem."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/11\/9275\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:42:14Z","timestamp":1760125334000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/11\/9275"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5,25]]},"references-count":53,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2023,6]]}},"alternative-id":["ijms24119275"],"URL":"https:\/\/doi.org\/10.3390\/ijms24119275","relation":{"has-preprint":[{"id-type":"doi","id":"10.26434\/chemrxiv-2023-r1cs3","asserted-by":"object"}]},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,5,25]]}}}