Tracking solvent and protein movement during CO ₂ release in carbonic anhydrase II crystals

Abstract

Carbonic anhydrases are mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO 2 /HCO 3 − . Previously, the X-ray crystal structures of CO 2 -bound holo (zinc-bound) and apo (zinc-free) human carbonic anhydrase IIs (hCA IIs) were captured at high resolution. Here, we present sequential timeframe structures of holo-[T = 0 s (CO 2 -bound), 50 s, 3 min, 10 min, 25 min, and 1 h] and apo-hCA IIs [T = 0 s, 50 s, 3 min, and 10 min] during the "slow" release of CO 2 . Two active site waters, W DW (deep water) and W DW ′ (this study), replace the vacated space created on CO 2 release, and another water, W I (intermediate water), is seen to translocate to the proton wire position W1. In addition, on the rim of the active site pocket, a water W2′ (this study), in close proximity to residue His64 and W2, gradually exits the active site, whereas His64 concurrently rotates from pointing away ("out") to pointing toward ("in") active site rotameric conformation. This study provides for the first time, to our knowledge, structural "snapshots" of hCA II intermediate states during the formation of the His64-mediated proton wire that is induced as CO 2 is released. Comparison of the holo-and apo-hCA II structures shows that the solvent network rearrangements require the presence of the zinc ion. − (general reviews are in refs. 1-5). In the hydration direction, the first step of catalysis is the conversion of CO 2 into bicarbonate through the nucleophilic attack of the reactive Znbound hydroxide, and the resultant bicarbonate is subsequently displaced from the zinc by a water molecule (expression 1) (6). The second step of catalysis is the transfer of a proton from the Zn-bound water to bulk solvent for the regeneration of the Znbound hydroxide. The general base for proton transfer (PT), B, can be either a proton acceptor in solution (water) or a residue (His64) in the enzyme (expression 2):

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

“…Such an interpretation ascertains that 7.0 atm CO 2 hCA II and 2.5 atm CO 2 hCA II are intermediate states that fill the gaps between the 15 atm pressurized CO 2 hCA II and the earliest time point of CO 2 release (15 atm CO 2 hCA II -50s) observed in the previous study (Kim et al, 2016). On this foundation, His64 and the water network near the active site were analyzed in order of decreasing internal CO 2 pressure (Fig.…”

“…2a and 3a; Kim et al, 2016). However, this W I disappeared in 15 atm CO 2 hCA II and W1 was observed to appear instead in the CO 2 -free 15 atm CO 2 hCA II -1h (as in Figs.…”

“…CO 2 entrapment using pressure cryocooling CO 2 entrapment was carried out as described in previous reports (Domsic et al, 2008;Kim et al, 2016). The hCA II crystals were first soaked in a cryosolution consisting of the reservoir solution supplemented with 20%(v/v) glycerol.…”

“…The refinement statistics are given in Table 1. We also re-refined the water molecules in the three previously reported structures (PDB entry 5dsi for 15 atm CO 2 hCA II, PDB entry 5dsj for 15 atm CO 2 hCA II -50s and PDB entry 5dsn for CO 2 hCA II -1h; Kim et al, 2016) is coloured cyan for clarity. Note that CO 2 is fully bound in the active site in (a) and (b).…”

“…These two structures were compared with the three previous structures (Kim et al, 2016) of hCA II crystals cryocooled under 15 atm CO 2 pressure and then incubated at room temperature for 0 s (1.2 Å resolution structure from PDB entry 5dsi; hereafter referred to as '15 atm CO 2 hCA II'), 50 s (1.25 Å resolution structure from PDB entry 5dsj; hereafter referred to as '15 atm CO 2 hCA II -50s') and 1 h (1.45 Å resolution structure from PDB entry 5dsn; the 'CO 2 -free state' and hereafter referred to as '15 atm CO 2 hCA II -1h'). The structural comparison reveals that 7.0 atm CO 2 hCA II and 2.5 atm CO 2 hCA II are previously unknown intermediate states between 15 atm CO 2 hCA II and 15 atm CO 2 hCA II -50 s. Together, these studies provide a view of how hCA II utilizes a water reservoir to fill the void in the active site as CO 2 is released.…”