Bicarbonate as a proton donor in catalysis by Zn(II)- and Co(II)-containing carbonic anhydrases.
Catalysis of (18)O exchange between CO(2) and water catalyzed by a Co(II)-substituted mutant of human carbonic anhydrase II is analyzed to show the rate of release of H(2)(18)O from the active site. This rate, measured by mass spectrometry, is dependent on proton transfer to the metal-bound (18)O-labeled hydroxide, and was observed in a site-specific mutant of carbonic anhydrase II in which a prominent proton shuttle residue His64 was replaced by alanine, which does not support proton transport. Upon increasing the concentration of bicarbonate, the rate of release of H(2)(18)O increased in a saturable manner to a maximum of 4 x 10(5) s(-)(1), consistent with proton transfer from bicarbonate to the Co(II)-bound hydroxide. The same mutant of carbonic anhydrase containing Zn(II) had the rate of release of H(2)(18)O smaller by 10-fold, but rate of interconversion of CO(2) and HCO(3)(-) about the same as the Co(II)-containing enzyme. These data as well as solvent hydrogen isotope effects suggest that the bicarbonate transferring the proton is bound to the cobalt in the enzyme. The enhancement of (18)O exchange caused by increasing bicarbonate concentration during catalysis by the Zn(II)-containing carbonic anhydrase from the archaeon Methanosarcina thermophila suggests that a very similar mechanism for proton donation by bicarbonate occurs with this wild-type enzyme.
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Departments of Pharmacology and Biochemistry, University of Florida College of Medicine, Gainesville, FL 32610-0267, USA.
Tripp
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BC
Ferry
J G
JG
Silverman
D N
DN
D019943:Amino Acid Substitution; D001639:Bicarbonates / Q000378:metabolism*; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010103:Oxygen Isotopes; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D001639:Bicarbonates; D007527:Isoenzymes; D010103:Oxygen Isotopes; D011994:Recombinant Proteins; D003300:Copper; D002256:Carbonic Anhydrases; D015032:Zinc