Journal:IUCrJ:S2052252520011008

Structural insights into the effect of active site mutation on carbonic anhydrase catalytic mechanism

Jin Kyun Kim, Cheol Lee, Seon Woo Lim, Jacob T. Andring, Aniruddha Adhikari, Robert McKenna and Chae Un Kim ^[1]

Molecular Tour
Enzymes greatly enhance catalytic rates and are therefore essential to speed up biochemical processes. The active sites of enzymes have highly optimized microenvironments for their specific substrates. Consequently, changes at the active site residues can have large effects on enzyme activity. However, direct prediction of a single mutation’s impact on an enzyme activity remains challenging due to the lack of precise correlations between the protein structure and its function at atomic resolution. In this study, we describe the effect of a single amino acid variant on a prototypical enzyme, human carbonic anhydrase II (CA II), by correlating its high-resolution reaction intermediate structures with the measured kinetic parameters.

Human carbonic anhydrases catalyze the reversible hydration/dehydration of CO₂/HCO₃-. In this study, we investigate Val143 to Ile (V143I) mutation that alters the hydrophobic pocket of the active site. V143I variant shows ~ 10 fold decrease in k_cat/K_M, while k_cat remains almost the same as the native CA II. Structural analysis was performed by comparing the catalytic intermediate states of native and V143I-CA II which were obtained by cryocooling protein crystals under 4 different CO₂ pressures (ranging from 0 (no CO₂ pressurization) to 15 atm). Structural changes in the CA II intermediates induced by the single residue mutation at the active site are identified. The V143I mutation in CA II produces steric hindrance and induces subtle changes in the active site electrostatic environment. The resulting effects on the CA II intermediates can be summarized as follows: (i) the dynamical motions and the allowed configurations of CO₂ are restricted, and the binding affinity of HCO₃- is increased with a distorted configuration, (ii) the water network in the water replenishment pathway is restructured, while (iii) the proton transfer dynamics is mostly unaffected. This detailed structural information can now be availed to assess the modifications in the reaction rate constants and the corresponding free energy profiles during the CO₂ hydration reaction of CA II.

. Overall protein backbones of the native and V143I CA II structures were very similar with the Cα – Cα r.m.s.d values being less than 0.14 Å and are represented as single white structure. The active site is located 15 Å deep from the surface. Note that Isoleucine 143 is located at the hydrophobic pocket in the active site. Active site of the V143I variant CA II 15 atm CO₂ is represented as red and native CA II 15 atm CO₂ is represented as white.

. The intermediate waters (WI and WI′) are colored in steel blue for clarity. .

Estimated free energy profiles for the CO₂ hydration reaction catalyzed by CA II. The energy states of the native CA II (black) are from previous study. The energy states of the V143I CA II (red) are qualitatively estimated with respect to the native form by considering the structural information and the variations in the reaction rate constants. Note that the energy level of [EZnH₂O + HCO₃-] in the V143I variant is assumed to be the same as in the native CA II. The depicted energy gaps are not to scale.

PDB references: human carbonic anhydrase II, native, 0 atm CO2, 6km3; 7 atm CO2, 6km4; 13 atm CO2, 6km5; 15 atm CO2, 6km6; V143I variant, 0 atm CO2, 6klz; 7 atm CO2, 6km0; 13 atm CO2, 6km1; 15 atm CO2, 6km2.

References

1. ↑ Kim JK, Lee C, Lim SW, Andring JT, Adhikari A, McKenna R, Kim CU. Structural insights into the effect of active-site mutation on the catalytic mechanism of carbonic anhydrase. IUCrJ. 2020 Sep 9;7(Pt 6):985-994. doi: 10.1107/S2052252520011008. eCollection, 2020 Nov 1. PMID:33209313 doi:http://dx.doi.org/10.1107/S2052252520011008