Buffer enhancement of proton transfer in catalysis by human carbonic anhydrase III.
Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.
Tu
C K
CK
Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville 32610.
Paranawithana
S R
SR
Jewell
D A
DA
Tanhauser
S M
SM
LoGrasso
P V
PV
Wynns
G C
GC
Laipis
P J
PJ
Silverman
D N
DN
D001665:Binding Sites; D002021:Buffers; D002245:Carbon Dioxide / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism
D002021:Buffers; D007093:Imidazoles; D011522:Protons; D011994:Recombinant Proteins; D002245:Carbon Dioxide; C029899:imidazole; D002256:Carbonic Anhydrases