title: Promiscuity of carbonic anhydrase II. Unexpected ester hydrolysis of carbohydrate-based sulfamate inhibitors.
abstract: Carbonic anhydrases (CAs) are enzymes whose endogenous reaction is the reversible hydration of CO(2) to give HCO(3)(-) and a proton. CA are also known to exhibit weak and promiscuous esterase activity toward activated esters. Here, we report a series of findings obtained with a set of CA inhibitors that showed quite unexpectedly that the compounds were both inhibitors of CO(2) hydration and substrates for the esterase activity of CA. The compounds comprised a monosaccharide core with the C-6 primary hydroxyl group derivatized as a sulfamate (for CA recognition). The remaining four sugar hydroxyl groups were acylated. Using protein X-ray crystallography, the crystal structures of human CA II in complex with four of the sulfamate inhibitors were obtained. As expected, the four structures displayed the canonical CA protein-sulfamate interactions. Unexpectedly, a free hydroxyl group was observed at the anomeric center (C-1) rather than the parent C-1 acyl group. In addition, this hydroxyl group is observed axial to the carbohydrate ring while in the parent structure it is equatorial. A mechanism is proposed that accounts for this inversion of stereochemistry. For three of the inhibitors, the acyl groups at C-2 or at C-2 and C-3 were also absent with hydroxyl groups observed in their place and retention of stereochemistry. With the use of electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry (ESI-FTICR-MS), we observed directly the sequential loss of all four acyl groups from one of the carbohydrate-based sulfamates. For this compound, the inhibitor and substrate binding mode were further analyzed using free energy calculations. These calculations suggested that the parent compound binds almost exclusively as a substrate. To conclude, we have demonstrated that acylated carbohydrate-based sulfamates are simultaneously inhibitor and substrate of human CA II. Our results suggest that, initially, the substrate binding mode dominates, but following hydrolysis, the ligand can also bind as a pure inhibitor thereby competing with the substrate binding mode.
authors.affiliation: Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Queensland 4111, Australia.
authors.forename: Marie
authors.initials: M
authors.lastname: Lopez
authors.forename: Hoan
authors.initials: H
authors.lastname: Vu
authors.forename: Conan K
authors.initials: CK
authors.lastname: Wang
authors.forename: Maarten G
authors.initials: MG
authors.lastname: Wolf
authors.forename: Gerrit
authors.initials: G
authors.lastname: Groenhof
authors.forename: Alessio
authors.initials: A
authors.lastname: Innocenti
authors.forename: Claudiu T
authors.initials: CT
authors.lastname: Supuran
authors.forename: Sally-Ann
authors.initials: SA
authors.lastname: Poulsen
mesh_terms: D002241:Carbohydrates / Q000737:chemistry*; D024402:Carbonic Anhydrase II / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004952:Esters / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D008968:Molecular Conformation; D013329:Structure-Activity Relationship; D013451:Sulfonic Acids / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D002241:Carbohydrates; D002257:Carbonic Anhydrase Inhibitors; D004952:Esters; D013451:Sulfonic Acids; C005741:sulfamic acid; D024402:Carbonic Anhydrase II