Abstract
Human carbonic anhydrase II (hCA II) is a zinc metalloenzyme that catalyzes the reversible hydration/dehydration of CO2/HCO3-. Although hCA II has been extensively studied to investigate the proton-transfer process that occurs in the active site, its underlying mechanism is still not fully understood. Here, ultrahigh-resolution crystallographic structures of hCA II cryocooled under CO2 pressures of 7.0 and 2.5 atm are presented. The structures reveal new intermediate solvent states of hCA II that provide crystallographic snapshots during the restoration of the proton-transfer water network in the active site. Specifically, a new intermediate water (WI') is observed next to the previously observed intermediate water WI, and they are both stabilized by the five water molecules at the entrance to the active site (the entrance conduit). Based on these structures, a water network-restructuring mechanism is proposed, which takes place at the active site after the nucleophilic attack of OH- on CO2. This mechanism explains how the zinc-bound water (WZn) and W1 are replenished, which are directly responsible for the reconnection of the His64-mediated proton-transfer water network. This study provides the first 'physical' glimpse of how a water reservoir flows into the hCA II active site during its catalytic activity.
Highlights
The reversible interconversion of carbon dioxide (CO2) and water to bicarbonate and a proton (H+) occurs at a rate that is limited by the diffusion of substrates in the presence of carbonic anhydrases (CAs) as the catalyst (Davenport, 1984; Christianson & Fierke, 1996; Chegwidden & Carter, 2000; Frost & McKenna, 2013; Supuran & De Simone, 2015)
In 15 atm CO2 Human carbonic anhydrase II (hCA II) – 50s, the electron density for the CO2 binding site is further shifted towards Zn and WZn, which correlates with the known positions of WDW and WD0 W (Fig. 2d)
In 15 atm CO2 hCA II – 1h, the electron density of the CO2 binding site splits into two distinct lobes, indicating that the CO2 site is completely replaced by WDW and WD0 W (Fig. 2e)
Summary
The reversible interconversion of carbon dioxide (CO2) and water to bicarbonate and a proton (H+) occurs at a rate that is limited by the diffusion of substrates in the presence of carbonic anhydrases (CAs) as the catalyst (Davenport, 1984; Christianson & Fierke, 1996; Chegwidden & Carter, 2000; Frost & McKenna, 2013; Supuran & De Simone, 2015). The general base (B) for the proton transfer is likely to be mediated by ordered waters and His within the enzyme, where the hydrophilic side of the active site forms the hydrogen-bonded water network (W1, W2, W20, W3a and W3b) that connects WZn to His64 This hydrogen-bonded network is believed to act as a proton wire that reduces the work required to transfer a proton from WZn to the bulk solvent for the regeneration of the zinc-bound OHÀ (2) (Silverman & McKenna, 2007; Steiner et al, 1975; Cui & Karplus, 2003; Fisher, Tu et al, 2007; Zheng et al, 2008; Fisher, Maupin et al, 2007; Silverman et al, 1979). The proton transfer during the catalytic activity is thought to occur via His through the proton wire rather than through the open entrance conduit
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
