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 kcat/KM, while kcat 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.
References
- ↑ 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
