Dataset Information

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

ABSTRACT: Carbonic anhydrases (CAs) catalyze the reversible hydration of CO(2) to HCO(3)(-) 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."

SUBMITTER: Becker HM

PROVIDER: S-EPMC3041061 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Becker Holger M HM Klier Michael M Schüler Christina C McKenna Robert R Deitmer Joachim W JW

Proceedings of the National Academy of Sciences of the United States of America 20110131 7

Carbonic anhydrases (CAs) catalyze the reversible hydration of CO(2) to HCO(3)(-) 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 h ...[more]

PMID: 21282642

Similar Datasets

Project description:Catalysis by the zinc metalloenzyme human carbonic anhydrase II (HCA II) is limited in maximal velocity by proton transfer between His64 and the zinc-bound solvent molecule. Asn62 extends into the active site cavity of HCA II adjacent to His64 and has been shown to be one of several hydrophilic residues participating in a hydrogen-bonded solvent network within the active site. We compared several site-specific mutants of HCA II with replacements at position 62 (Ala, Val, Leu, Thr, and Asp). The efficiency of catalysis in the hydration of CO 2 for the resulting mutants has been characterized by (18)O exchange, and the structures of the mutants have been determined by X-ray crystallography to 1.5-1.7 A resolution. Each of these mutants maintained the ordered water structure observed by X-ray crystallography in the active site cavity of wild-type HCA II; hence, this water structure was not a variable in comparing with wild type the activities of mutants at residue 62. Crystal structures of wild-type and N62T HCA II showed both an inward and outward orientation of the side chain of His64; however, other mutants in this study showed predominantly inward (N62A, N62V, N62L) or predominantly outward (N62D) orientations of His64. A significant role of Asn62 in HCA II is to permit two conformations of the side chain of His64, the inward and outward, that contributes to maximal efficiency of proton transfer between the active site and solution. The site-specific mutant N62D had a mainly outward orientation of His64, yet the difference in p K a between the proton donor His64 and zinc-bound hydroxide was near zero, as in wild-type HCA II. The rate of proton transfer in catalysis by N62D HCA II was 5% that of wild type, showing that His64 mainly in the outward orientation is associated with inefficient proton transfer compared with His64 in wild type which shows both inward and outward orientations. These results emphasize the roles of the residues of the hydrophilic side of the active site cavity in maintaining efficient catalysis by carbonic anhydrase.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets