Enhancement of the catalytic properties of human carbonic anhydrase III by site-directed mutagenesis

Abstract

Among the seven known isozymes of carbonic anhydrase in higher vertebrates, isozyme III is the least efficient in catalytic hydration of CO2 and the least susceptible to inhibition by sulfonamides. We have investigated the role of two basic residues near the active site of human carbonic anhydrase III (HCA III), lysine 64 and arginine 67, to determine whether they can account for some of the unique properties of this isozyme. Site-directed mutagenesis was used to replace these residues with histidine 64 and asparagine 67, the amino acids present at the corresponding positions of HCA II, the most efficient of the carbonic anhydrase isozymes. Catalysis by wild-type HCA III and mutants was determined from the initial velocity of hydration of CO2 at steady state by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and water at chemical equilibrium by mass spectrometry. We have shown that histidine 64 functions as a proton shuttle in carbonic anhydrase by substituting histidine for lysine 64 in HCA III. The enhanced CO2 hydration activity and pH profile of the resulting mutant support this role for histidine 64 in the catalytic mechanism and suggest an approach that may be useful in investigating the mechanistic roles of active-site residues in other isozyme groups. Replacing arginine 67 in HCA III by asparagine enhanced catalysis of CO2 hydration 3-fold compared with that of wild-type HCA III, and the pH profile of the resulting mutant was consistent with a proton transfer role for lysine 64. Neither replacement enhanced the weak inhibition of HCA III by acetazolamide or the catalytic hydrolysis of 4-nitrophenyl acetate.

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein−Ligand Binding

“…652 18 refolded with similar rates (within a factor of 2) and yield from the SDS-denatured state. This study demonstrates that modifying the charge on the surface of BCA II does not the affect the ability of the protein to refold.…”

“…Figure 14B shows simulated curves for the pHdependence of the affinity of arylsulfonamides for CA II: the product of fractions of CA II and arylsulfonamide in the active forms specified in eqs [16][17][18][19]. This figure also shows experimental data for the pH-dependence of for the complex between HCA II and pnitrobenzenesulfonamide (3).…”

“…Silverman estimated the activity of HCA II and BCA II in intact cells using mass spectrometry to detect the exchange of 18 O between CO 2 and water at equilibrium. 361 Alternatively, 13 C NMR can assess the activity of CA by measuring the exchange of 13 Table 8 provides a summary of techniques used for measuring binding constants of CA to various ligands.…”

Inhibition of Carbonic Anhydrase II by Thioxolone: A Mechanistic and Structural Study

“…It is well-known that different CA isozymes vary greatly in their ability to cleave esters; for example, CA III has very little esterase activity with 4-NPA compared to CA II (94). Because this type of prodrug inhibitor mechanism depends on activation by enzymatic cleavage of ester bonds, this class of inhibitor may have advantages in determining isozyme specificity.…”

Expression, Assay, and Structure of the Extracellular Domain of Murine Carbonic Anhydrase XIV

“…CA XIV also contains the proton shuttle residue His-64, which is present in all enzymes with high activity. Because proton release is the rate-limiting step in catalysis (46,47), isozymes without this residue display substantially lower activity (48). In CA XIV, His-64 adopts the "out" conformer (data not shown).…”