Production of Active Human Carbonic Anhydrase II in E. Coli.

Forsman, Cecilia; Behravan, Gity; Osterman, A.L.; Jonsson, Bengt‐Harald; Enzell, Curt R.; Berg, Jan-Eric; Bartók, Mihály; Pelczer, István; Dombi, György

doi:10.3891/acta.chem.scand.42b-0314

Cited by 81 publications

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“…hCA II protein was expressed, purified, and crystallized as described previously (22,(33)(34)(35). The CO 2 entrapment inside hCA II crystals was carried out by cryocooling crystals under CO 2 pressure of 15 atm (19,20).…”

Section: Methodsmentioning

confidence: 99%

Tracking solvent and protein movement during CO ₂ release in carbonic anhydrase II crystals

Kim

Song

Avvaru

et al. 2016

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

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Carbonic anhydrases are mostly zinc metalloenzymes that catalyze the reversible hydration/dehydration of CO 2 /HCO 3 − . Previously, the X-ray crystal structures of CO 2 -bound holo (zinc-bound) and apo (zinc-free) human carbonic anhydrase IIs (hCA IIs) were captured at high resolution. Here, we present sequential timeframe structures of holo-[T = 0 s (CO 2 -bound), 50 s, 3 min, 10 min, 25 min, and 1 h] and apo-hCA IIs [T = 0 s, 50 s, 3 min, and 10 min] during the "slow" release of CO 2 . Two active site waters, W DW (deep water) and W DW ′ (this study), replace the vacated space created on CO 2 release, and another water, W I (intermediate water), is seen to translocate to the proton wire position W1. In addition, on the rim of the active site pocket, a water W2′ (this study), in close proximity to residue His64 and W2, gradually exits the active site, whereas His64 concurrently rotates from pointing away ("out") to pointing toward ("in") active site rotameric conformation. This study provides for the first time, to our knowledge, structural "snapshots" of hCA II intermediate states during the formation of the His64-mediated proton wire that is induced as CO 2 is released. Comparison of the holo-and apo-hCA II structures shows that the solvent network rearrangements require the presence of the zinc ion. − (general reviews are in refs. 1-5). In the hydration direction, the first step of catalysis is the conversion of CO 2 into bicarbonate through the nucleophilic attack of the reactive Znbound hydroxide, and the resultant bicarbonate is subsequently displaced from the zinc by a water molecule (expression 1) (6). The second step of catalysis is the transfer of a proton from the Zn-bound water to bulk solvent for the regeneration of the Znbound hydroxide. The general base for proton transfer (PT), B, can be either a proton acceptor in solution (water) or a residue (His64) in the enzyme (expression 2):

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Section: Methodsmentioning

confidence: 99%

Tracking solvent and protein movement during CO ₂ release in carbonic anhydrase II crystals

Kim

Song

Avvaru

et al. 2016

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

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“…Protein Purification and Crystallization-hCAII was expressed in a recombinant strain of Eshcherichia coli (BL21(DE3)pLysS) containing a plasmid encoding the hCAII gene (14). Purification was carried out via affinity chromatography as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II

Domsic

Avvaru

Kim

et al. 2008

Journal of Biological Chemistry

214

206

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The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO 2 -pressurized, cryo-cooled crystals to capture the first step of CO 2 hydration catalyzed by the zincmetalloenzyme human carbonic anhydrase II, the binding of substrate CO 2 , for both the holo and the apo (without zinc) enzyme to 1.1 Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO 2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675-3679). These structures provide insight into the long hypothesized binding of CO 2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO 2 . This method may also have a much broader implication for the study of other enzymes for which CO 2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.Since their discovery (2), the carbonic anhydrases (CAs) 3 have been extensively studied because of their important physiological functions in all kingdoms of life (3). This family of enzymes is broadly comprised of three well studied, structurally distinct families (␣, ␤, and ␥) of mostly zinc-metalloenzymes that catalyze the reversible hydration of CO 2 to bicarbonate (3, 4). More recently there have been other more distinct CAs characterized, such as a cadmium ␤ class-mimic CA (5). However, all appear to share the same overall catalytic mechanism composed of two independent stages, shown in Equations 1 and 2, an example of a ping-pong mechanism (6, 7). In the hydration direction, the first stage is the conversion of CO 2 into bicarbonate via a nucleophilic attack on CO 2 by the reactive zinc-bound hydroxide. The resultant bicarbonate is then displaced from the zinc by a water molecule (Reaction 1).The second stage is the transfer of a proton from the zincbound water to bulk solvent to regenerate the zinc-bound hydroxide (Reaction 2). Here B is a proton acceptor in solution or a residue of the enzyme itself.For hCAII (␣ class CA), this reaction is facilitated by a solvent molecule with a pK a near 7 that is directly coordinated to the zinc (6). This centrally located zinc exhibits a tetrahedral configuration with three histidines (His-94, His-96, and His-119) and either a water or a hydroxide molecule. The active site cavity can be loosely described as being conical in shape having a 15 Å diameter entrance that tapers into the center of the enzyme. The cavity is partitioned into two very different environments. On one side of the zinc, deep within the active site, lies a cluster of hydrophobic amino acids (namely , whereas on the other side of the zinc, leading out of the active site to the bulk solvent, the surface is lined with hydrophilic amino acids (namely Tyr-7, Asn-62...

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“…Mutants were identified by dideoxy sequencing [8] of randomly picked clones. The mutant DNA was subcloned into an expression plasmid described previously [9]. After transfection, the mutated human carbonic anhydrases I1 were expressed in the lonEscherichia coli strain SG20043.…”

Section: Materials and Methods Enzymementioning

confidence: 99%

Fine tuning of the catalytic properties of human carbonic anhydrase II

Behravan

Jonsson

Lindskog

1991

European Journal of Biochemistry

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The active‐site residue Thr200 in human carbonic anhydrase II has been replaced by several different amino acids by site‐directed mutagenesis. The CO2 hydration and 4‐nitrophenyl acetate hydrolase activities of these variants have been measured, as well as inhibition by the monovalent anion, SCN−. The results show that the replacement of Thr200 with Ser or Ala has no significant effect on the catalyzed rates of CO2 hydration. Also, variants with Asn200 and Gly200 have high activities, whereas the activities of variants with Val, Ile or Arg at position 200 are reduced by factors of 2–3 compared to the unmodified enzyme. The variant with Asp200 has a very low activity in both reactions studied, while most of the other variants have enhanced esterase activities, Thr200↔ Arg isoenzyme II as much as sevenfold. The Asp200 variant has a low affinity for SCN− as well as for a sulfonamide inhibitor, whereas all the other variants bind SCN− more strongly than unmodified enzyme. While His200 characterizes carbonic anhydrases I, the presence of Arg, Val or Ile as well as His at position 200 in human isoenzyme II seems to result in isoenzyme‐I‐like functional properties.

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Production of Active Human Carbonic Anhydrase II in E. Coli.

Cited by 81 publications

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Tracking solvent and protein movement during CO ₂ release in carbonic anhydrase II crystals

Tracking solvent and protein movement during CO ₂ release in carbonic anhydrase II crystals

Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II

Fine tuning of the catalytic properties of human carbonic anhydrase II

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Production of Active Human Carbonic Anhydrase II in E. Coli.

Cited by 81 publications

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Tracking solvent and protein movement during CO 2 release in carbonic anhydrase II crystals

Tracking solvent and protein movement during CO 2 release in carbonic anhydrase II crystals

Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II

Fine tuning of the catalytic properties of human carbonic anhydrase II

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Tracking solvent and protein movement during CO ₂ release in carbonic anhydrase II crystals

Tracking solvent and protein movement during CO ₂ release in carbonic anhydrase II crystals