Intramolecular proton shuttle supports not only catalytic but also noncatalytic function of carbonic anhydrase II

Carbonic anhydrases (CAs) catalyze the reversible hydration of CO₂ to HCO₃⁻ and H⁺. The rate-limiting step in this reaction is the shuttle of protons between the catalytic center of the enzyme and the bulk solution. In carbonic anhydrase II (CAII), the fastest and most wide-spread isoform, this H⁺ s...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2011-02, Vol.108 (7), p.3071-3076
Main Authors: Becker, Holger M, Klier, Michael, Schüler, Christina, McKenna, Robert, Deitmer, Joachim W
Format: Article
Language:English
Subjects:
Acidification
Animals
Bicarbonates - metabolism
Biochemistry
Biological Sciences
Biological Transport - physiology
Blotting, Western
Carbon Dioxide - metabolism
Carbonic Anhydrase II - chemistry
Carbonic Anhydrase II - metabolism
Carbonic Anhydrase III - chemistry
Carbonic Anhydrase III - metabolism
Catalysis
Catalytic activity
Cell membranes
Cells
Enzymes
Frogs
Gene expression
Humans
Hydrogen-Ion Concentration
Imidazoles
Lactates
Lactic Acid - metabolism
Models, Molecular
Molecular structure
Monocarboxylic Acid Transporters - metabolism
Oocytes
Oocytes - metabolism
Protein isoforms
Proteins
Protons
Rates of change
Xenopus
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Summary:Carbonic anhydrases (CAs) catalyze the reversible hydration of CO₂ to HCO₃⁻ and H⁺. The rate-limiting step in this reaction is the shuttle of protons between the catalytic center of the enzyme and the bulk solution. In carbonic anhydrase II (CAII), the fastest and most wide-spread isoform, this H⁺ shuttle is facilitated by the side chain of His64, whereas CA isoforms such as carbonic anhydrase III (CAIII), which lack such a shuttle, have only low catalytic activity in vitro. By using heterologous protein expression in Xenopus oocytes, we tested the role of this intramolecular H⁺ shuttle on CA activity in an intact cell. The data revealed that CAIII, shown in vitro to have ~1,000-fold reduced activity as compared with CAII, displays significant catalytic activity in the intact cell. Furthermore, we tested the hypothesis that the H⁺ shuttle in CAII itself can facilitate transport activity of the monocarboxylate transporters 1 and 4 (MCT1/4) independent of catalytic activity. Our results show that His64 is essential for the enhancement of lactate transport via MCT1/4, because a mutation of this residue to alanine (CAII-H64A) abolishes the CAII-induced increase in MCT1/4 activity. However, injection of 4-methylimidazole, which acts as an exogenous H⁺ donor/acceptor, can restore the ability of CAII-H64A to enhance transport activity of MCT1/4. These findings support the hypothesis that the H⁺ shuttle in CAII not only facilitates CAII catalytic activity but also can enhance activity of acid-/base-transporting proteins such as MCT1/4 in a direct, noncatalytic manner, possibly by acting as an "H⁺-collecting antenna."
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1014293108