Structural and Kinetic Analysis of the Chemical Rescue of the Proton Transfer Function of Carbonic Anhydrase II

Duda, David M.; Tu, Chingkuang; Qian, Minzhang; Laipis, Philip J.; Agbandje‐McKenna, Mavis; Silverman, David N.; McKenna, Robert

doi:10.1021/bi002295z

Cited by 104 publications

(156 citation statements)

References 22 publications

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“…The importance of His64 for intramolecular H + shuttling and CAII catalytic activity has been demonstrated by a site-specific mutant in which His64 is replaced by alanine (CAII-H64A), which caused an ∼20-fold decrease in the H + transfer rate (1). However, addition of exogenous H + donors/ acceptors, usually derivates of carnosine (β-analyl-L-histidine) or imidazole [e.g., 4-methylimadazole (4-MI)], can recover the activity of CAII-H64A almost fully to the wild-type level, whereas other buffers, such as Hepes or 3-(N-morpholino)propanesulfonic acid, cannot restore H + shuttling in CAII-H64A (1,4). Crystallographic studies revealed that the primary binding site for 4-MI within the CAII molecule is at the rim of the active-site cavity, where 4-MI π-stacks with the indole ring of Trp5 (5).…”

mentioning

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.

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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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mentioning

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.

Self Cite

118

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“…Imidazole and imidazole derivatives mimic the PSR function of His-64 and rescue the H64A variant of CA-II that is 10-fold reduced in k cat . Crystal structures of the variant complexed with 4-methylimidazole show the rescue agent occupying the "out" position leading to the conclusion that this orientation of His-64 is important for proton transfer (18). On the other hand, aqueous phase molecular dynamics simulations of the wild-type enzyme in three protonation states indicate that His-64 primarily assumes the "in" orientation, a result leading the authors to suggest that fluctuations between the two orientations of this residue may have limited influence on proton transfer (17).…”

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confidence: 99%

Carbonic Anhydrase: New Insights for an Ancient Enzyme

Tripp¹,

Smith²,

Ferry

2001

Journal of Biological Chemistry

503

393

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“…Thus, this scheme explains the effective proton release following the intra-molecular proton transfer step in the catalytic reaction of hCAII. This scheme can be also used to explain the unique pH-dependent activity (9,56,57) of this enzyme, which has its maximum activity in pH 7. First, lowering pH accelerates that His 64 would not participate in the hydrogen-bonded pathway because this residue takes the out conformation at low pH regions as shown in Fig.…”

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confidence: 99%

Tautomerism of Histidine 64 Associated with Proton Transfer in Catalysis of Carbonic Anhydrase

Shimahara

Yoshida

Shibata

et al. 2007

Journal of Biological Chemistry

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. These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His 64 can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time-or energy-consuming processes.Carbonic anhydrase (CA) 2 (EC 4.2.1.1) is a ubiquitous enzyme that catalyzes the reversible hydration of carbon dioxide (1). Isozymes of carbonic anhydrase regulate or function in such diverse physiological processes as pH regulation, ion transport, water-electrolyte balance, bicarbonate secretion-absorption, bone resorption, maintenance of intraocular pressure, renal acidification, and brain development (2). Nonfunctioning CA is implicated in such diseases as osteopetrosis syndrome, glaucoma, respiratory acidosis, epilepsy, and Méni-ère syndrome. Diseases due to CA deficiency include those affecting bones, the brain, and the kidneys. Consequently determining the detailed structure/function relationships or mechanisms responsible for its catalytic properties is mandatory for developing inhibitors or replacement therapies.CA is present in at least three gene families (␣, ␤, and ␥), which has made it a popular model for the study of the evolution of gene families and protein folding, and for transgenic and gene target studies (2). Among the three families, the ␣ family is the best characterized, with 11 known isozymes identified in mammals. Earnhardt and co-workers have summarized maximal k cat and k cat /K m values for CO 2 hydration by isozyme I-VII (3). The human isozyme II (hCAII) has a remarkably high turnover rate or catalytic efficiency (k cat /K m ϭ 1.5 ϫ 10 8 M Ϫ1 s Ϫ1 ) that is very close to the frequency with which the enzyme and substrate molecules collide with each other in solution.It is widely accepted that the hydration of CO 2 catalyzed by hCAII proceeds through several chemical steps as shown in Scheme 1 (1, 4, 5): the direct nucleophilic attack of the zinc-bound hydroxide ion on the carbonyl carbon of substrate CO 2 (structures 1-2), the formation of a zinc-bound bicarbonate intermediate (structures 2-3), the isomerization of the bicarbonate ion (structures 3-4), the exchange of the product bicarbonate ion with a H 2 O (structures 4 -5), and the regeneration of the zinc-bound hydroxide ion by the transfer of a proton to bulk solvent (structures 1-5). The proton transfer step (structures 1-5) consists of two substeps: 1) an intra-molecular transfer of protons to another residue in the enzyme and 2) a release of protons to the outside of the enzyme with the aid of a base. The intra-molecular proton transfer is the rate-limiting step of the maximal turnover rate (10 6 s Ϫ1 ) at high concentrations of a base, whereas the proton release into the medium is rate-limiting at low buffer concentrations.

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Structural and Kinetic Analysis of the Chemical Rescue of the Proton Transfer Function of Carbonic Anhydrase II

Cited by 104 publications

References 22 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

Carbonic Anhydrase: New Insights for an Ancient Enzyme

Tautomerism of Histidine 64 Associated with Proton Transfer in Catalysis of Carbonic Anhydrase

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