title: Cloning, expression, purification and sulfonamide inhibition profile of the complete domain of the η-carbonic anhydrase from Plasmodium falciparum.
abstract: We report the cloning, purification and characterization of the full domain of carbonic anhydrase (CA, EC 4.2.1.1) from Plasmodium falciparum, which incorporates 358 amino acid residues (from 181 to 538, in the sequence of this 600 amino acid long protein), called PfCAdom. The enzyme, which belongs to the η-CA class showed the following kinetic parameters: kcat of 3.8×10(5)s(-1) and kcat/Km of 7.2×10(7)M(-1)×s(-1), being 13.3 times more effective as a catalyst compared to the truncated form PfCA. PfCAdom is more effective than the human (h) isoform hCA I, being around 50% less effective compared to hCA II, one of the most catalytically efficient enzymes known so far. Intriguingly, the sulfonamides CA inhibitors generally showed much weaker inhibitory activity against PfCAdom compared to PfCA, prompting us to hypothesize that the 69 amino acid residues insertion present in the active site of this η-CA is crucial for the active site architecture. The best sulfonamide inhibitors for PfCAdom were acetazolamide, methazolamide, metanilamide and sulfanilamide, with KIs in the range of 366-808nM.
authors.affiliation: Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, Napoli, Italy; Università degliStudi di Firenze, Dipartimento Di Chimica, Laboratorio di ChimicaBioinorganica, Polo Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Del Prete
authors.affiliation: Università degliStudi di Firenze, Dipartimento Di Chimica, Laboratorio di ChimicaBioinorganica, Polo Scientifico, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Vullo
authors.affiliation: Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, Napoli, Italy.
authors.forename: Viviana
authors.initials: V
authors.lastname: De Luca
authors.affiliation: Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, Napoli, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Carginale
authors.affiliation: King Saud University, Department of Chemistry, Riyadh, Saudi Arabia.
authors.forename: Sameh M
authors.initials: SM
authors.lastname: Osman
authors.affiliation: King Saud University, Department of Chemistry, Riyadh, Saudi Arabia.
authors.forename: Zeid
authors.initials: Z
authors.lastname: AlOthman
authors.affiliation: King Saud University, Department of Chemistry, Riyadh, Saudi Arabia; Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy. Electronic address: claudiu.supuran@unifi.it.
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.affiliation: Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, Napoli, Italy. Electronic address: clemente.capasso@ibbr.cnr.it.
authors.forename: Clemente
authors.initials: C
authors.lastname: Capasso
mesh_terms: D000595:Amino Acid Sequence; D024401:Carbonic Anhydrase I / Q000037:antagonists & inhibitors / Q000378:metabolism; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology*; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000737:chemistry* / Q000494:pharmacology
chemical_list: D002257:Carbonic Anhydrase Inhibitors; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013449:Sulfonamides; D024401:Carbonic Anhydrase I; D024402:Carbonic Anhydrase II; D002256:Carbonic Anhydrases
keywords: Carbonic anhydrase; Hydratase activity; Inhibitors; Malaria; Metalloenzymes; Protozoa; Sulfonamide