Revisiting Zinc Coordination in Human Carbonic Anhydrase II

Abstract

Carbonic anhydrase (CA) is a well-studied, zinc-dependent metalloenzyme that catalyzes the hydrolysis of carbon dioxide to the bicarbonate ion. The apo-form of CA (apoCA) is relatively easy to generate, and the reconstitution of the human erythrocyte CA has been initially investigated. In the past, these studies have continually relied on equilibrium dialysis measurements to ascertain an extremely strong association constant (Ka ~ 1.2×1012) for Zn2+. However, new reactivity data and isothermal titration calorimetry (ITC) data reported herein call that number into question. As shown in the ITC experiments, the catalytic site binds a stoichiometric quantity of Zn2+ with a strong equilibrium constant (Ka ~ 2 × 109) that is three orders of magnitude lower than the previously established value. The thermodynamic parameters associated with Zn2+ binding to apoCA are unraveled from a series of complex equilibria associated with the in vitro metal binding event. This in-depth analysis adds clarity to the complex ion chemistry associated with zinc binding to carbonic anhydrase and validates thermochemical methods that accurately measure association constants and thermodynamic parameters for complex-ion and coordination chemistry observed in vitro. Additionally, the zinc sites in both the as-isolated and reconstituted ZnCA were probed using X-ray absorption spectroscopy. Both X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses indicate the zinc center in the reconstituted carbonic anhydrase is nearly identical to that of the as-isolated protein and confirms the notion that the metal binding data reported herein is the reconstitution of the zinc active site of human CA II.

Dissecting ITC data of metal ions binding to ligands and proteins

“…Since ligand pKa values are not known, the total number of protons that are displaced upon metal binding (a + 2b in Scheme 1) cannot be calculated using the approach described above. An alternate method [73,74], which takes advantage of the change in ΔH HB if the experimental buffer is altered, has been widely used in ITC experiments [4,10,11,24,26,41,75]. Practically, this strategy requires multiple titrations to be carried out at the same pH in at least three different buffers under otherwise identical experimental conditions.…”

“…As such, only indirect experiments that rely on competition with another metal ion or spectroscopic probe can be used to investigate Zn 2+ -protein interactions. Alternatively, Zn 2+ binding events are characterized by a quantifiable heat signature which can be exploited by ITC to gain insight into Zn 2 + -dependent systems such as zinc finger domains [4][5][6][7], carbonic anhydrase [8][9][10], and insulin [11]. Additionally, ITC has played a role in understanding countless important bioinorganic systems including ferritin and transferrin [12][13][14][15][16][17], amyloid beta [18,19], nucleases [20][21][22][23] and cardiac troponin C [24].…”

A De Novo Designed Metalloenzyme for the Hydration of CO2

“…These affinities are weaker than that of CAII measured by equilibrium dialysis (K d = 0.8 ± 0.1 pM); [16] however, recent isothermal titration calorimetry experiments suggest a three-orders of magnitude weaker affinity for CAII (K d = 0.45 nM). [17] Based on this newer measurement, α 3 DH 3 has an affinity only two-orders of magnitude weaker than CAII and stronger than those observed for our previously published Hg(II) S Zn(II) N (TRIL9CL23H) 3 (K d = 0.8 ± 0.1 and 0.22 ± 0.06 μM at pH 7.5 and 9.0, respectively). [18] A sample of Zn(II)α 3 DH 3 at pH 9.0 was analyzed by extended x-ray absorption fine structure (EXAFS) spectroscopy.…”

“…These parameters are very similar to EXAFS distances measured for CAII (Zn-N/O of 1.98 Å). [17] The true indication of a successfully designed metalloenzyme mimic is catalytic activity toward the physiological reaction of the natural enzyme, in this case, the hydration of CO 2 . Using Khalif's [19] stopped flow indicator technique, Zn(II)α 3 DH 3 was found to be an efficient catalyst with activities that increase with pH (Table 2).…”

Sculpting Metal‐binding Environments in De Novo Designed Three‐helix Bundles

“…Human carbonic anhydrase II is one of the most efficient enzymes (approaching the diffusion limit) which catalyzes the reversible interconversion between CO 2 and HCO 3 −. [56] Even though the mechanism, structure, and inhibition have been previously studied, de novo protein design still offers a novel approach to study and replicate an important function of a native metalloenzyme in a simplified peptide system. We have previously demonstrated a carbonic anhydrase model in a bimetallic 3SCC construct [Hg(II)] S [Zn(II)(H 2 O/OH − )] N (TRIL9CL23H) 3 n + and Zastrow et al reported this model to be within 500-fold of the fastest isozyme (CAII), which is the fastest CA model to date (Figure 2a).…”