Structural and Kinetic Effects on Changes in the CO₂ Binding Pocket of Human Carbonic Anhydrase II

Abstract

This work examines the effect on catalysis of perturbing the position of bound CO2 in the active site of human carbonic anhydrase II (HCA II). Variants of HCA II replacing Val143 with hydrophobic residues, Ile, Leu, and Ala, were examined. The efficiency of catalysis in the hydration of CO2 for these variants was characterized by 18O exchange mass spectrometry, and their structures determined by X-ray crystallography at 1.7 to 1.5 Å resolution. The most hydrophobic substitutions V143I and V143L showed decreases in catalysis, as much as 20-fold, while the replacement by the smaller V143A showed only a moderate two-fold decrease in activity. Structural data for all three variants show no significant change in overall position of amino-acid side chains in the active site compared with wild type. However, V143A HCA II showed additional ordered water molecules in the active site compared to wild type. To further investigate the decrease in catalytic efficiency of V143I HCA II, an X-ray crystallographic CO2 entrapment experiment was performed to 0.93 Å resolution. This structure revealed an unexpected shift of the CO2 substrate towards the zinc bound solvent, placing it ~0.3 Ǻ closer than previously observed in wild type in conjunction with the observed dual occupancy of the product bicarbonate, presumably formed during the data acquisition. These data suggest that the Ile substitution at position 143 reduced catalytic efficiency is likely due to steric crowding resulting in destabilization of the transition state for conversion of CO2 into bicarbonate and a decreased product dissociation rate.

Structural Basis for the Inhibition of Helicobacter pylori α-Carbonic Anhydrase by Sulfonamides

“…However, there is no experimental evidence for the essential role of Thr200 in HCAII catalysis. The observation that the side chains of all residues that are implicated in the catalytic mechanism of HCAII are conserved in HpαCA and their positions and conformations are very close to those of respective residues in the human homologue strongly suggests that HpαCA is likely to follow the same reaction mechanism where CO 2 is converted into bicarbonate HCO 3 - via a nucleophilic attack on CO 2 by the reactive zinc-bound hydroxide ion [ 41 , 49 ].…”

Human carbonic anhydrase II–cyanate inhibitor complex: putting the debate to rest

“…This binding site is located near the backbone amide of Thr199 between the zinc and the hydrophobic pocket that includes Val121, Val143, Leu198 and Val207 (Hå kansson et al, 1992;Jö nsson et al, 1993). This hydrophobic region has been shown to be essential for the binding of the CO 2 substrate (Fierke et al, 1991;Nair et al, 1991;Krebs et al, 1993;Domsic et al, 2008;Sjö blom et al, 2009;West et al, 2012) and is highly conserved between -CA isozymes.…”

“…In addition, studies with CA II variants in which Val143 (a residue in close proximity to the CO 2 -binding site) was replaced by Ile, Leu or Ala showed a decrease in the catalytic rate and displacement of CO 2 binding compared with wild-type CA II (West et al, 2012). Hence, a ligand mimicking CO 2 binding would behave differently when comparing the wild type and the V207I CA II variant.…”

Cloning, purification and preliminary crystallographic analysis of the complex ofHelicobacter pyloriα-carbonic anhydrase with acetazolamide

“…H. pylori has CA genes from both the and families (Chirica et al, 2002). Periplasmic H. pylori CA (Hp CA) shares 28% sequence identity with human carbonic anhydrase II (Chirica et al, 2002) and is likely to follow the same reaction mechanism, in which CO 2 is converted into bicarbonate (HCO 3 À ) via a nucleophilic attack on CO 2 by the reactive zinc-bound hydroxide and the resultant bicarbonate is then displaced from the zinc by a water molecule (West et al, 2012;Lindskog, 1997 Krulwich et al, 2011).…”