Ultra-High Resolution X-Ray Diffraction from Crystals of the Kinetic Mutant of Human Carbonic Anhydrase II, His 64 Ala, and its Complexes with Proton Acceptor/Donors

Crystals of human carbonic anhydrase II with a specific point mutation, His 64 to Ala, have been grown in a solution of ammonium sulfate in the presence of mercury chloride. The crystals appear in approximately two weeks and belong to the monoclinic space group P21, with unit cell parameters of a = 42.2 Å, b = 41.4 Å, c = 71.9 Å, β = 104.2 and one carbonic anhydrase molecule in the asymmetric unit. The crystals diffract X-rays beyond 1.0 Å resolution. These crystals, soaked with exogenous proton acceptor/donors, will be used in X-ray and neutron diffraction studies to map the fine water structure “proton wire” in the active site of carbonic anhydrase and to assign the intraand intermolecular proton transfer pathway(s) from the zinc-bound water out to the bulk solvent. Introduction Carbonic anhydrase II (CA II), one of the most efficient of the isozymes in the α class of carbonic anhydrases [1], catalyzes the hydration of CO2 in two stages [2,3]. The first is the conversion of CO2 into bicarbonate by reaction with the zinc-bound hydroxide; the dissociation of bicarbonate leaves a water molecule bound to the zinc (Eq. 1). [H2O] CO2 + EZnOH EZnHCO3 EZnH2O + HCO3 (1) EZnH2O + B EZnOH + BH (2) The second step is the transfer of a proton to solution to regenerate the zinc-bound hydroxide (Eq. 2); here B designates a proton acceptor, either an exogenous proton acceptor from solution or a residue of the enzyme itself, although ultimately the proton must be transported to bulk solvent. A considerable number of studies have shown that His 64 in human CA II (HCA II) functions as a proton shuttle in this manner [4-6]. A site-specific mutant of HCA II in which His64 is replaced with Ala (H64A HCA II), has been shown to decrease the rate of the proton transfer step (Eq. 2) by more than 10-fold compared with wild type HCA II [7]. This loss of catalytic activity of H64A HCA II can be rescued by 4-methylimidazole (4-MI), an exogenous proton acceptor/donor (Fig. 1), in a saturable process with a maximum activity of 40% of wild-type HCA II [7]. The crystal structure of the rescued complex at 1.6 Å resolution shows 4-MI bound in the active-site cavity of H64A HCA II, through π stacking interactions with residue Trp5 and H-bonding interactions with water molecules [7]. Crystals of H64A HCA II that diffract X-rays beyond 1 Å resolution have been grown to study the fine structural changes in the activite site associated with the loss and rescue by exogenous acceptor/proton donors (Fig. 1) of the proton transfer step (Eq. 2). Because of the exceptionally high quality of these crystals, the structures obtained from X-ray and neutron diffraction studies can be used to elucidate the detailed structure of intervening water channel(s) “proton wire(s)” by which a proton can be transferred from the zinc-bound hydroxide to the bulk solvent (Eq. 2). Preliminary experiments conducted at the Laue diffraction (LADI) experimental station at the Institut Laue-Langevin (ILL) confirms the feasibility of low temperature (12K) neutron crystallography of these crystals. Figure 1. Chemical structure of exogenous acceptor/proton donors of proton shuttling in Human Carbonic Anhydrase Mutant His64Ala. Methods Expression and purification: H64A HCA II was prepared and expressed in E. coli as described in previous publications [5,8] and purified by affinity chromatography [9]. The mutation was confirmed by sequencing the DNA of the entire coding region for H64A HCA II in the expression vector. Crystallization and X-ray diffraction data collection: Crystals of H64A HCA II (Fig. 2) were produced by the hanging drop method [10], using a 10 mg/ml enzyme solution in 50 mM Tris·HCl, pH = 7.8, containing 1 N N