Histidine 64 is not required for high CO<sub>2</sub> hydration activity of human carbonic anhydrase II

Forsman, Cecilia; Behravan, Gity; Jonsson, Bengt‐Harald; Liang, Zhiwei; Lindskog, Sven; Ren, Xilin; Sandström, Johan; Wallgren, Katarina

doi:10.1016/0014-5793(88)81156-6

Cited by 52 publications

(36 citation statements)

References 8 publications

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“…Proton transfer in CAII is mediated by the imidazole ring in His64, and replacement of His64 by alanine leads to a significant loss of CAII enzymatic activity in vitro in the absence of exogenous buffers (1). However, addition of certain buffers such as carnosine, imidazole, and methylimidazole can restore catalytic function in vitro (1,22,24). We now have analyzed catalytic activity of CAII-H64A in an intact cell.…”

Section: Discussionmentioning

confidence: 99%

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

Becker¹,

Klier

Schüler

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

118

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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.”

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Section: Discussionmentioning

confidence: 99%

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

Becker¹,

Klier

Schüler

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

118

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“…The importance of His 64 in intramolecular proton transfer was recently called into question by the findings from the site-specific mutagenesis studies on His 64 of CA 11 [62]. The results show that mutations from His 64 to lysine, glutamine, glutamic acid, and alanine lead to changes of k,,, values of CO, hydration by 1.5-3.5-fold as compared with the value in the native enzyme at pH 8.8 and 25°C.…”

Section: Displacement Of Zn2+-bound Hco With a Water Moleculementioning

confidence: 99%

Theoretical study of carbonic anhydrase‐catalyzed hydration of co₂: A brief review

Liang

Lipscomb

1989

Int J of Quantum Chemistry

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Quantum mechanical calculations have been used to study the reaction mechanism of human carbonic anhydrase-catalyzed hydration of CO,. This reaction is responsible for fast metabolism of CO, in the human body. For each of the reaction steps, possible catalytic effects of active site residues are examined. The pertinent results are as follows.(1) For COz binding, the experimentally observed 2.5 cm-' frequency shift of the asymmetic stretching frequency between measurements taken in the aqueous solution and in the enzyme is reproduced in our theoretical calculations. Our results suggest that CO, binds to the zinc ion within the hydrophobic pocket. (2) No energy barrier is found for the nucleophilic attack from Zn2+-bound OH-to C of COz to form Znz'-bound HCO;. (3) For the internal proton transfer within zinc-bound HCO;, the barrier of 35.6 kcaI/mol for the direct internal proton transfer is reduced to 3.5 and 1.4 kcal/mol, respectively, when one or two water molecules are included for proton relay. (4) Displacement of ZnZ+-bound HCOj by H,O is facilitated by the presence of the negatively charged Glu 106-Thr 199 chain and by the association and the subsequent ionization of a fifth water ligand. ( 5 ) For the intramolecular proton transfer between Znz+-bound H 2 0 and His 64, the ZnZ+ ion lowers the pK, of Zn2+-bound water and repels the proton. His 64, or a similar proton receptor with a larger proton affinity than H,O, functions as proton receiver; and the active site water molecules visualized by x-ray crystallography are important for the proton relay function. In summary, it is demonstrated that in order to achieve effective catalysis, a sequence of precisely coordinated catalytic events among all participating catalytic elements in the enzyme's active site is essential.

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“…Residue 64 is a histidine, except in CA 111 where it is a lysine. However, this does not explain the low activity of CA 111, as Forsman et al (1988) have used sitedirected mutagenesis to replace His 64 of CA I1 with Lys (or Gln, Glu, Ala) and found a fall in activity of only 1.5-3.5-fold-not the 67-fold fall expected for a CA 111 like enzyme. Residue 67 could be more important for high activity with Asn in CA 11 and Gln in CA VI (both high-activity isozymes), while the lower activity CA I has His and the very low activity CA 111 Arg in this position.…”

Section: Discussionmentioning

confidence: 94%

Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

Aldred¹,

Fu²,

Barrett³

et al. 1991

Biochemistry

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Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CA VI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension [Fernley, R. T., Wright, R. D., & Coghlan, J. P. (1988b) Biochemistry 27, 2815]. Overall the human CA VI protein has a sequence identity of 35% with human CA II, while residues involved in the active site of the enzymes have been conserved. The human sheep secreted carbonic anhydrases have a sequence identity of 72%. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

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Histidine 64 is not required for high CO₂ hydration activity of human carbonic anhydrase II

Cited by 52 publications

References 8 publications

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

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

Theoretical study of carbonic anhydrase‐catalyzed hydration of co₂: A brief review

Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

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Histidine 64 is not required for high CO2 hydration activity of human carbonic anhydrase II

Cited by 52 publications

References 8 publications

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

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

Theoretical study of carbonic anhydrase‐catalyzed hydration of co2: A brief review

Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

Contact Info

Product

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Histidine 64 is not required for high CO₂ hydration activity of human carbonic anhydrase II

Theoretical study of carbonic anhydrase‐catalyzed hydration of co₂: A brief review