Roles of the conserved aspartate and arginine in the catalytic mechanism of an archaeal β-class carbonic anhydrase

The roles of an aspartate and an arginine, which are completely conserved in the active sites of β-class carbonic anhydrases, were investigated by steady-state kinetic analyses of replacement variants of the β-class enzyme (Cab) from the archaeon Methanobacterium thermoautotrophicum. Previous kinetic analyses of wild-type Cab indicated a two-step zinc-hydroxide mechanism of catalysis in which the k_cat/K_m value depends only on the rate constants for the CO₂ hydration step, whereas k_cat also depends on rate constants from the proton transfer step (K. S. Smith, N. J. Cosper, C. Stalhandske, R. A. Scott, and J. G. Ferry, J. Bacteriol. 182:6605-6613, 2000). The recently solved crystal structure of Cab shows the presence of a buffer molecule within hydrogen bonding distance of Asp-34, implying a role for this residue in the proton transport step (P. Strop, K. S. Smith, T. M. Iverson, J. G. Ferry, and D. C. Rees, J. Biol. Chem. 276:10299-10305, 2001). The k_cat/K_m values of Asp-34 variants were decreased relative to those of the wild type, although not to an extent which supports an essential role for this residue in the CO₂ hydration step. Parallel decreases in k_cat and k_cat/K_m values for the variants precluded any conclusions regarding a role for Asp-34 in the proton transfer step; however, the k_cat of the D34A variant was chemically rescued by replacement of 2-(N-morpholino)propanesulfonic acid buffer with imidazole at pH 7.2, supporting a role for the conserved aspartate in the proton transfer step. The crystal structure of Cab also shows Arg-36 with two hydrogen bonds to Asp-34. Arg-36 variants had both k_cat and k_cat/K_m values that were decreased at least 250-fold relative to those of the wild type, establishing an essential function for this residue. Imidazole was unable to rescue the k_cat of the R36A variant; however, partial rescue of the kinetic parameter was obtained with guanidine-HCl indicating that the guanido group of this residue is important.