Active-site solvent replenishment observed during human carbonic anhydrase II catalysis

Abstract

-site solvent replenishment.PDB references: 2.5 atm CO 2 hCA II, 5y2r; 7 atm CO 2 hCA II, 5y2s; 15 atm CO 2 hCA II, re-refined, 5yui; 15 atm CO 2 hCA II -50s, re-refined, 5yuj; 15 atm CO 2 hCA II -1h, re-refined, 5yuk . 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 CO 2 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 (W I 0 ) is observed next to the previously observed intermediate water W I , 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 À on CO 2 . This mechanism explains how the zinc-bound water (W Zn ) 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.

Carbonic anhydrase under pressure

“…An article in the current issue of IUCrJ describes crystallographic analyses of hCAII at ultrahigh resolution where the crystals were exposed to CO 2 under 7 and 2.5 atm pressure (Kim et al, 2018). These studies extend previous studies where the crystals were exposed to 15 atm pressure of CO 2 and frozen to liquid nitrogen temperature after various lengths of time (Kim et al, 2016).…”

Carbonic anhydrase II microcrystals suitable for XFEL studies

“…Although this reaction has been extensively studied and X-ray crystal structures of CA II in complex with substrate and product have been elucidated, there is still little structural information regarding the dynamics of the catalytic mechanism (Domsic et al, 2008;Domsic & McKenna, 2010;Kim et al, 2016Kim et al, , 2018Fierke et al, 1991). Hence, the use of pump-probe, time-resolved SFX (TR-SFX) would be the preferred methodology to visualize the pathways of substrate/ product entry/exit from the active site.…”

Structure and mechanism of copper–carbonic anhydrase II: a nitrite reductase

“…While the origin of these ligands is unknown, others have hypothesized synchrotron radiation as a possible source of high-energy ions leading to the formation of these molecules (Fukuda et al, 2015). One of the NO 2 À oxygens occupies the position of the Zn-CAII deep water, which is important for solvent replenishment (Kim et al, 2016(Kim et al, , 2018. Comparison of the Zn-CAII:CO 2 complex (PDB entry 5yui, Kim et al, 2016) with the Cu-CAII:NO 2 À complex shows significant differences.…”