The functional role of the highly conserved active site Arg 59 in the prototype of the γ-class carbonic anhydrase Cam (carbonic anhydrase from Methanosarcina thermophila) was investigated. Variants (R59A, -C, -E, -H, -K, -M, and -Q) were prepared by site-directed mutagenesis and characterized by size exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, and stopped-flow kinetic analyses. CD spectra indicated similar secondary structures for the wild type and the R59A and -K variants, independent of nondenaturing concentrations of guanidine hydrochloride (GdnHCl). SEC indicated that all variants purified as homotrimers like the wild type. SEC also revealed that the R59A and -K variants unfolded at ≥ 1.5 M GdnHCl, compared to 3.0 M GdnHCl for the wild type. These results indicate that Arg 59 contributes to the thermodynamic stability of the Cam trimer. The R59K variant had kcat and kcat/Km values that were 8 and 5% of the wild-type values, respectively, while all other variants had kcat and kcat/Km values 10-100-fold lower than those of the wild type. The R59A, -C, -E, -M, and -Q variants exhibited 4-63-fold increases in kcat and 9-120-fold increases in kcat/Km upon addition of 100 mM GdnHCl, with the largest increases observed for the R59A variant, which was comparable to the R59K variant. The kinetic results indicate that a positive charge at position 59 is essential for the CO2 hydration step of the overall catalytic mechanism.
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