Rate-equilibria relationships in intramolecular proton transfer in human carbonic anhydrase III.
Maximal turnover rates for the dehydration of HCO3- catalyzed by the zinc metalloenzyme carbonic anhydrase III are limited by a proton transfer to zinc-bound hydroxide in the active site. We have used site-directed mutagenesis to place a proton donor, histidine, at position 64 and used 18O exchange between CO2 and water measured by mass spectrometry to determine the rates of intramolecular proton transfer to the zinc-bound hydroxide. In a series of site-specific mutants, the values of pKa of the zinc-bound water ranged from approximately 5 to 9. The rate constants for proton transfer obeyed a Brønsted correlation and showed sharp curvature characteristic of facile proton transfers. Application of Marcus rate theory shows that this proton transfer has the small intrinsic energy barrier (near 1.5 kcal/mol) characteristic of rapid proton transfer between nitrogen and oxygen acids and bases, but has an observed overall energy barrier (near 10 kcal/mol), indicating the involvement of accompanying, energy requiring processes such as solvent reorganization or conformational change.
Silverman
D N
DN
Department of Pharmacology and Therapeutics, University of Florida, Gainesville 32610.
Tu
C
C
Chen
X
X
Tanhauser
S M
SM
Kresge
A J
AJ
Laipis
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism
D001639:Bicarbonates; D007527:Isoenzymes; D011522:Protons; D011994:Recombinant Proteins; D002256:Carbonic Anhydrases; D015032:Zinc