New Model for a Theoretical Density Functional Theory Investigation of the Mechanism of the Carbonic Anhydrase:  How Does the Internal Bicarbonate Rearrangement Occur?

Bottoni, Andreà; Lanza, Camilla Zaira; Miscione, Gian Pietro; Spinelli, Domenico

doi:10.1021/ja030336j

Cited by 70 publications

(87 citation statements)

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“…1). A wealth of indirect evidence indicates that the deep water Wat-338 position serves as the primary substrate-binding site (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), although the atomic details of enzyme and substrate-product interaction have remained elusive until now.…”

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confidence: 99%

Structural study of X-ray induced activation of carbonic anhydrase

Sjöblom

Polentarutti

Djinović-Carugo

2009

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

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Carbonic anhydrase, a zinc metalloenzyme, catalyzes the reversible hydration of carbon dioxide to bicarbonate. It is involved in processes connected with acid-base homeostasis, respiration, and photosynthesis. More than 100 distinct human carbonic anhydrase II (HCAII) 3D structures have been generated in last 3 decades [ CO 2 binding ͉ crystal pressurization ͉ radiation driven reaction ͉ substrate-product complex structure T he HCAII enzyme is a functional 29-kDa monomer consisting of a 10-stranded, twisted ␤-sheet. The active site is located at the bottom of a 15-Å cone-shaped cavity that leads to the center of the protein (37). Key features of the active site ( Fig. 1) include a zinc ion coordinated tetrahedrally by 3 histidine residues (His-94, His-96, and His-119) and a water molecule/ hydroxide ion as a fourth ligand (Wat-263).Thr-199, a key residue of the second coordination sphere, is important for enzyme activity; together with Thr-200, it is involved in a finely tuned network of hydrogen bonds leading toward the solvent-exposed His-64, which is located at the entrance of the active-site channel. Thr-199 forms a hydrogen bond to the zinc-bound water/hydroxide (Wat-263), thereby orienting the 2 lone hydroxide electron pairs toward the 2 neighboring water molecules (Wat-318 and Wat-338) that reside on potential substrate-binding sites. Although both positions are suitable for a nucleophilic attack of the zinc-bound hydroxide ion, their environments differ substantially.Wat-318 is located in a hydrophilic environment on the way out of the active-site cone, whereas the ''deep water'' Wat-338 is located in a hydrophobic pocket that is lined by the following side chains: (Fig. 1). A wealth of indirect evidence indicates that the deep water Wat-338 position serves as the primary substrate-binding site (1-12), although the atomic details of enzyme and substrate-product interaction have remained elusive until now.The generally accepted catalytic mechanism of carbonic anhydrase (13) is described by a 3-step kinetic scheme: (i) a Zn-OH Ϫ moiety catalyzes the interconversion of CO 2 to HCO 3 Ϫ , leaving a water molecule as the fourth zinc ligand (Eq. 1); (ii) a proton is then transferred from the zinc-bound water to the imidazole ring of His-64 (Eq. 2); and (iii) this proton then leaves His-64 for the surrounding solvent (Eq. 3).The pK a values for both the zinc-bound water and the proton shuttle (His-64) are close to 7. Xenon under pressure was for the first time used in structural biology employing either solution NMR or single-crystal X-ray diffraction methods to probe cavities in protein structures (14,15). The crystal pressurization with noble gases, such as xenon or krypton, was subsequently recognized as a successful heavy atom derivatization method to solve the phase problem in macromolecular crystallography (16). We used this method for functional studies of an enzyme-substrate complex, similarly to at the bottom of the active site. Wat-318 is in a hydrophilic environment toward the mouth of the activ...

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confidence: 99%

Structural study of X-ray induced activation of carbonic anhydrase

Sjöblom

Polentarutti

Djinović-Carugo

2009

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

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“…52 The return to the zinc-water resting state is achieved by the rearrangement of the linked bicarbonate followed by its displacement by a water molecule. 28,49,53,54 While there is no X-ray structure of HCA II with HCO 3 À bound to zinc, crystallographic studies of a Thr200His mutant of HCA II 55 We suggest that this fine-tuning coordination mode could be correlated with the proton transfer from Glu117 to His119. Indeed, from a Zn 2+ -ImH 0 Á Á ÁMeCOO À unit, the shuttling of the proton between the H-bonded atoms of the Glu117-His119 couple will decrease the zinc charge and thus its affinity to bicarbonate which will concomitantly shift to a monodentate coordination to zinc.…”

Section: Relevance Of Proton Transfer To the Hca II Catalytic Cyclementioning

confidence: 86%

Metal-histidine-glutamate as a regulator of enzymatic cycles: a case study of carbonic anhydrase

Frison¹,

Ohanessian²

2009

Phys. Chem. Chem. Phys.

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To cite this version:Gilles Frison, Gilles Ohanessian. Metal-histidine-glutamate as a regulator of enzymatic cycles: a case study of carbonic anhydrase.. Physical Chemistry Chemical Physics, Royal Society of Chemistry, 2009Chemistry, , 11 (2), pp.374-383. 10.1039 Metal-histidine-glutamate as a regulator of enzymatic cycles: a case study of carbonic anhydrasew Gilles Frison* and Gilles Ohanessian Histidine is a very common metal ligand in metalloenzymes. Besides being an efficient Lewis base, its electronic properties are essential to shape the metal ability to catalyze the reaction. Here we show that histidine's properties can be tuned, in turn, by an easy proton transfer to a nearby glutamate. We study this situation in Human Carbonic Anhydrase II (HCA II) in which one of the three histidines bound to zinc (His119) interacts also with a glutamate residue (Glu117). Proton transfer from His119 to Glu117 has been hypothesized in the past, however realistic modeling is performed here for the first time. We show that the carboxylate group of Glu117 behaves only as a hydrogen bond acceptor in the hydroxy form of HCA II. On the other hand, our results suggest that Glu117 could exist either as a hydrogen bond acceptor or as a proton acceptor in the aqua form of HCA II, the two isomers having almost the same thermodynamic stability. We propose that this proton shift may be used by the enzyme to facilitate the final displacement of bicarbonate by water.

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“…8E, a and b the reaction (Scheme 1) makes it reasonable that the water bridge ( Fig. 1) can split in a process such as isomerization of a zinc-bound bicarbonate ion (54,55) or the exchange of the product bicarbonate ion with a water molecule in the reaction. This indicates that a flow of the water molecules should occur in the active site to continue the reaction.…”

Section: ⑀2mentioning

confidence: 99%

“…9, C and D). In this step, we adopted the Lipscomb model for isomerization of the bicarbonate ion on the zinc ion according to the recent papers (54,55). However, this does not necessarily give it any preference to the Linskog model; our scheme might not depend on the isomerization mechanism of the bicarbonate ion.…”

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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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New Model for a Theoretical Density Functional Theory Investigation of the Mechanism of the Carbonic Anhydrase: How Does the Internal Bicarbonate Rearrangement Occur?

Cited by 70 publications

References 46 publications

Structural study of X-ray induced activation of carbonic anhydrase

Structural study of X-ray induced activation of carbonic anhydrase

Metal-histidine-glutamate as a regulator of enzymatic cycles: a case study of carbonic anhydrase

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

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