Bicarbonate as a proton donor in catalysis by Zn(II)- and Co(II)-containing carbonic anhydrases

C. Tu, B. C. Tripp, James Gregory Ferry, D. N. Silverman

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Catalysis of18O exchange between CO2 and water catalyzed by a Co(II)-substituted mutant of human carbonic anhydrase II is analyzed to show the rate of release of H218O from the active site. This rate, measured by mass spectrometry, is dependent on proton transfer to the metal-bound 18O-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 H218O increased in a saturable manner to a maximum of 4 × 105 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 H218O smaller by 10-fold, but rate of interconversion of CO2 and HCO3- 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 18O 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.

Original languageEnglish (US)
Pages (from-to)5861-5866
Number of pages6
JournalJournal of the American Chemical Society
Volume123
Issue number25
DOIs
StatePublished - Oct 3 2001

Fingerprint

Carbonic anhydrase
Carbonic Anhydrase II
Carbonic Anhydrases
Bicarbonates
Catalysis
Protons
Proton transfer
Enzymes
Ion exchange
Methanosarcina
Isotopes
Mass spectrometry
Cobalt
Alanine
Archaea
Hydrogen
Metals
Mass Spectrometry
Catalytic Domain
Water

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

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title = "Bicarbonate as a proton donor in catalysis by Zn(II)- and Co(II)-containing carbonic anhydrases",
abstract = "Catalysis of18O exchange between CO2 and water catalyzed by a Co(II)-substituted mutant of human carbonic anhydrase II is analyzed to show the rate of release of H218O from the active site. This rate, measured by mass spectrometry, is dependent on proton transfer to the metal-bound 18O-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 H218O increased in a saturable manner to a maximum of 4 × 105 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 H218O smaller by 10-fold, but rate of interconversion of CO2 and HCO3- 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 18O 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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Bicarbonate as a proton donor in catalysis by Zn(II)- and Co(II)-containing carbonic anhydrases. / Tu, C.; Tripp, B. C.; Ferry, James Gregory; Silverman, D. N.

In: Journal of the American Chemical Society, Vol. 123, No. 25, 03.10.2001, p. 5861-5866.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Bicarbonate as a proton donor in catalysis by Zn(II)- and Co(II)-containing carbonic anhydrases

AU - Tu, C.

AU - Tripp, B. C.

AU - Ferry, James Gregory

AU - Silverman, D. N.

PY - 2001/10/3

Y1 - 2001/10/3

N2 - Catalysis of18O exchange between CO2 and water catalyzed by a Co(II)-substituted mutant of human carbonic anhydrase II is analyzed to show the rate of release of H218O from the active site. This rate, measured by mass spectrometry, is dependent on proton transfer to the metal-bound 18O-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 H218O increased in a saturable manner to a maximum of 4 × 105 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 H218O smaller by 10-fold, but rate of interconversion of CO2 and HCO3- 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 18O 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.

AB - Catalysis of18O exchange between CO2 and water catalyzed by a Co(II)-substituted mutant of human carbonic anhydrase II is analyzed to show the rate of release of H218O from the active site. This rate, measured by mass spectrometry, is dependent on proton transfer to the metal-bound 18O-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 H218O increased in a saturable manner to a maximum of 4 × 105 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 H218O smaller by 10-fold, but rate of interconversion of CO2 and HCO3- 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 18O 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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