Abstract
We have prepared a site-specific mutant of human carbonic anhydrase (HCA) II with histidine residues at positions 7 and 64 in the active site cavity. Using a different isozyme, we have placed histidine residues in HCA III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His(7) in HCA III. Using an (18)O exchange method to measure rate constants for intramolecular proton transfer, we have found that inserting two histidine residues into the active site cavity of either isozyme II or III of carbonic anhydrase results in rates of proton transfer to the zinc-bound hydroxide that are antagonistic or suppressive with respect to the corresponding single mutants. The crystal structure of Y7H HCA II, which contains both His(7) and His(64) within the active site cavity, shows the conformation of the side chain of His(64) moved from its position in the wild type and hydrogen-bonded through an intervening water molecule with the side chain of His(7). This suggests a cause of decreased proton transfer in catalysis.
Highlights
The carbonic anhydrases in the ␣ class include the mammalian isozymes and are all zinc-containing monomeric enzymes, generally with molecular masses near 30 kDa
We have placed histidine residues in human carbonic anhydrase (HCA) III at positions 64 and 67 and in another mutant at positions 64 and 7. Each of these histidine residues can act as a proton transfer group in catalysis when it is the only nonliganding histidine in the active site cavity, except His[7] in HCA III
This was initially determined by observation of a solvent deuterium isotope effect of 3.8 on kcat for hydration catalyzed by human carbonic anhydrase (HCA)[1] II, among the most efficient isozymes in the ␣ class of carbonic anhydrases (3)
Summary
The carbonic anhydrases in the ␣ class include the mammalian isozymes and are all zinc-containing monomeric enzymes, generally with molecular masses near 30 kDa. The maximal velocities of catalysis and the rates of exchange of 18O between CO2 and water are limited by the intramolecular proton transfers indicated in Equation 2. This was initially determined by observation of a solvent deuterium isotope effect of 3.8 on kcat for hydration catalyzed by human carbonic anhydrase (HCA)[1] II, among the most efficient isozymes in the ␣ class of carbonic anhydrases (3). Subsequent results including pH profiles, buffer activation, and computer simulations for these isozymes support the rate-limiting nature of proton transfer in these isozymes (4 –7)
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