Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the β- and γ-class

Chingkuang Tu, Roger S. Rowlett, Brian C. Tripp, James G. Ferry, David N. Silverman

Research output: Contribution to journalArticlepeer-review

39 Scopus citations

Abstract

Catalysis of the dehydration of HCO3- by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide. Carbonic anhydrases in each of the α, β, and γ classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the α class. We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (β class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (γ class). A series of structurally homologous imidazole and pyridine buffers were used as proton acceptors in the activation of CO2 hydration at steady state and as proton donors of the exchange of 18O between CO2 and water at chemical equilibrium. Free energy plots of the rate constants for this intermolecular proton transfer as a function of the difference in pKa of donor and acceptor showed extensive curvature, indicating a small intrinsic kinetic barrier for the proton transfers. Application of Marcus rate theory allowed quantitative estimates of the intrinsic kinetic barrier which were near 0.3 kcal/mol with work functions in the range of 7-11 kcal/mol for mutants in the β and γ class, similar to results obtained for mutants of carbonic anhydrase in the α class. The low values of the intrinsic kinetic barrier for all three classes of carbonic anhydrase reflect proton transfer processes that are consistent with a model of very rapid proton transfer through a flexible matrix of hydrogen-bonded solvent structures sequestered within the active sites of the carbonic anhydrases.

Original languageEnglish (US)
Pages (from-to)15429-15435
Number of pages7
JournalBiochemistry
Volume41
Issue number51
DOIs
StatePublished - Dec 24 2002

All Science Journal Classification (ASJC) codes

  • Biochemistry

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