Effect of disease-linked mutations on the structure, function, stability and aggregation of human carbonic anhydrase II.

Point mutations in gene sequence often lead to protein misfolding or destabilization which is a well-known cause of a number of loss-of-function diseases. The carriers of point mutations in the human carbonic anhydrase II (HCAII) gene have been recognized to display carbonic anhydrase II deficiency syndrome (CADS). Two such single point mutations linked with CADS involve Gly145Arg and His94Tyr substitution. In the present study, we obtained these two single mutants of HCAII using site-directed mutagenesis, and successfully expressed and purified them. To examine the effect of mutations on the structure and function of HCAII, we carried out circular dichroism, intrinsic fluorescence, NMR measurements and activity assays. Studies suggest that the mutant proteins undergo local structural perturbations and have compromised native state stability. HCAIIH94Y (H94Y), being an active site mutant, shows larger destabilization effect as compared to HCAIIG145R (G145R). GdnHCl-denaturation studies showed that HCAII unfolding is a two-step process (N ⇌ I ⇌ U) and the free energy of first transition (N ⇌ I) decreases by 1.5 kJ mol-1 and 4.9 kJ mol-1 for G145R and H94Y, respectively. Conformational changes and enzyme activity were established through various spectroscopic techniques.