Human Carbonic Anhydrase. Protein Conformation and Metal Ion Binding<sup>*</sup>

Coleman, Joseph E.

doi:10.1021/bi00888a014

Cited by 121 publications

(49 citation statements)

References 36 publications

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“…Such comparatively rapid reactions have been reported with Zn-MT and Zn-finger proteins [9,23,28,29]. But Zn-enzymes such as carboxypeptidase, superoxide dismutase, and carbonic anhydrase appear much more sluggish in their metal ion exchange chemistry [30–32]. …”

Section: Discussionmentioning

confidence: 93%

Toxic metal proteomics: Reaction of the mammalian zinc proteome with Cd2+

Namdarghanbari

Bertling

Krezoski

et al. 2014

Journal of Inorganic Biochemistry

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The hypothesis was tested that Cd2+ undergoes measureable reaction with the Zn-proteome through metal ion exchange chemistry. The Zn-proteome of pig kidney LLCPK1 cells is relatively inert to reaction with competing ligands, including Zinquin acid, EDTA, and apo-metallothionein. Upon reaction of Cd2+ with the Zn-proteome, Cd2+ associates with the proteome and near stoichiometric amounts of Zn2+ become reactive with these chelating agents. The results strongly support the hypothesis that Cd2+ displaces Zn2+ from native proteomic binding sites resulting in the formation of a Cd-proteome. Mobilized Zn2+ becomes adventitiously bound to proteome and available for reaction with added metal binding ligands. Cd-proteome and Zn-metallothionein readily exchange metal ions, raising the possibility that this reaction restores functionality to Cd-proteins. In a parallel experiment, cells were exposed to Cd2+ and pyrithione briefly to generate substantial proteome-bound Cd2+. Upon transition to a Cd2+ free medium, the cells generated new metallothionein protein over time that bound most of the proteomic Cd2+ as well as additional Zn2+.

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Section: Discussionmentioning

confidence: 93%

Toxic metal proteomics: Reaction of the mammalian zinc proteome with Cd2+

Namdarghanbari

Bertling

Krezoski

et al. 2014

Journal of Inorganic Biochemistry

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“…Another possibility is that the pH-dependence is the result of a cluster of interacting groups which act more or less as a unit [8].…”

Section: Discussionmentioning

confidence: 99%

Inhibition and Modification of Human Carbonic Anhydrase B with Bromoacetate and Iodoacetamide

Whitney

1970

European Journal of Biochemistry

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1. Bromoacetate and iodoacetamide reversibly inhibit human erythrocyte carbonic anhydrase B. The binding of these inhibitors is competitive with that of the other inhibitors, chloride and sulfanilamide. However, bromoacetate and iodoacetamide are only partially competitive with each other. That is, both can bind a t the same time, but the binding of one inhibitor increases the Ki for the second by about 50°/,.2. While bound as a reversible inhibitor, bromoacetate or iodoacetamide reacts with the basic form o f a specific histidine residue in the enzyme. The half-times of reaction are 0.44 h and 2.7 h, respectively. The pH-dependence of reaction shows that the pK of this histidine is 5.6 when bromoacetate is bound as a reversible inhibitor, and the pK when iodoacetamide is bound is below 5.0. 3.Reaction with bromoacetate introduces a carboxymethyl group in the region of the active center. This group perturbs the active site of the enzyme. This specific carboxymethylation causes about a five-fold decrease in esterase activity and a shift in the midpoint of the pH-dependence curve of activity from about pH 7 . 3 to 9.2. The affinity for several inhibitors is decreased. 4.Results of titration of the carboxymethyl enzyme with acetazolamide are consistent with 1.0 binding sites per enzyme molecule. This is the same number of sites as for native enzyme, but the Ki is higher with modified enzyme.5 . Studies of the visible spectrum of cobaltous carbonic anhydrase B have revealed two pH-dependent transitions. The first occurs below pH 7 and produces relatively small changes in the spectrum. Introduction of the carboxymethyl group has little effect on the first transition, but shifts the midpoint of the second transition from pH 7.1 to 9.2.6. Several lines of evidence indicate that the carboxymethyl group in the modified enzyme occupies a position different from the bromoacetate ion in the reversible complex.Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide. (For a review of this subject, see Maren [I].) However, the enzyme will also catalyze other reactions, such as the dehydration of aldehydes [a] and the hydrolysis of esters [3-51. The latter activity is very useful because of the simplicity of the assay.Carbonic anhydrase contains one mole of Zn2f which is essential for activity. The metal can be removed and activity regenerated by addition of Zn2+ or Co2+. The cobaltous enzyme has a visible spectrum which is sensitive to changes in pH and to the addition of inhibitors [6--81. Reaction with these reagents introduces a perturbing group in the region of the active center. Reaction causes complete inactivation in the case of the substituted sulfonamide and over 80°/, inactivation with the other reagents. These reagents react with human carbonic anhydrase A in the same way as with

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“…The catalytic activity of carbonic anhydrase is linked to one ionizing group in the active site with pK around 7 [20,. The nature of this group is not known, but it might be, for example, an amino acid side chain such as imidazole or a metal-coordinated water molecule [15,20,35]. This group with an apparent p K of 7.2 and 7.5 at 0.05 and 0.15 M KC14, respectively, can easily be fitted to the titration data.…”

Section: _ _ ~mentioning

confidence: 99%

Hydrogen Ion Equilibria and the Chemical Modification of Lysine and Tyrosine Residues in Bovine Carbonic Anhydrase B

Nilsson

Lindskog

1967

Eur J Biochem

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1. Potentiometric titrations of bovine carbonic anhydrase B have been performed. On titration with acid from the isoionic point, pH 5.65, seven histidines, which are masked in the native enzyme, become protonated during a conformational change occurring in a narrow pH-range near pH 4. Concomitantly, the enzyme activity is lost. On back-titration of the acid-denatured enzyme from pH 2, all 11 histidines are titratable. The denatured enzyme shows an increase of titratable groups in the alkaline pH-range as well.2. Spectrophotometric titrations a t alkaline p H show that most of the tyrosines are masked in the native enzyme. Only one tyrosine seems to titrate freely but with a rather high value of the intrinsic pK (10.8). The remaining seven tyrosines ionize above pH 12 during a time-dependent change leading to activity loss.3. Tetranitromethane reacts with only one tyrosine in the native enzyme. The modified enzyme retains full catalytic activity. 4.Methyl acetimidate reacts with a t least 95 of the lysines, including the carboxyl-terminal residue, without loss of activity. The number of titratable groups in the pH region 9 to I1 is accordingly decreased. Several forms of carbonic anhydrase have been isolated from human and bovine erythrocytes [l-41.These enzyme forms have quite similar properties, but marked differences in details have been found, including differences in catalytic behavior [2,5]. All forms contain one zinc ion which is essential for activity [6 -81, and they have about the same moleccular weight [3,9,10]. The amino acid compositions of the human forms B and C and the bovine form B, are considerably different [3,9,11], but it has recently been shown that there are some sequences common for two or all three forms [12]. The results of measurements of optical rotatory dispersion [13-161 and circular dichroism [I31 indicate common features but also distinct differences in the conformations of the various enzyme forms.We have continued the comparative approach to the problem of the structure-activity relationship in carbonic anhydrase and present in this paper the results of studies on the hydrogen ion equilibria of the bovine enzyme, form B. We have found that the titration behavior of this form differs from those of the human forms B and C [17,18] as expected from the differences in amino acid compositions and, in addition, that a larger number of tyrosyl residues seem to be masked in the bovine enzyme. Most of the histidyl residues are masked in the native states of Enzyme. Carbonic anhydrase or carbonate hydro-lyase (EC 4.2.1.1).both the human and bovine forms and become protonized only after denaturation in acid. MATERIALS AND METHODS Bovine Erythrocyte Curbonic Anhydruse BThis enzyme was prepared as previously described by Lindskog [l]. The final zone electrophoresis step was omitted, however, and replaced by rechromatography on a DEAE-cellulose column (d, 4 c m ; 1, 40 cm). This procedure gave a higher yield of form B than the original method, and the enzyme had the same degree of purity as jud...

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Human Carbonic Anhydrase. Protein Conformation and Metal Ion Binding^*

Cited by 121 publications

References 36 publications

Toxic metal proteomics: Reaction of the mammalian zinc proteome with Cd2+

Toxic metal proteomics: Reaction of the mammalian zinc proteome with Cd2+

Inhibition and Modification of Human Carbonic Anhydrase B with Bromoacetate and Iodoacetamide

Hydrogen Ion Equilibria and the Chemical Modification of Lysine and Tyrosine Residues in Bovine Carbonic Anhydrase B

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Human Carbonic Anhydrase. Protein Conformation and Metal Ion Binding*

Cited by 121 publications

References 36 publications

Toxic metal proteomics: Reaction of the mammalian zinc proteome with Cd2+

Toxic metal proteomics: Reaction of the mammalian zinc proteome with Cd2+

Inhibition and Modification of Human Carbonic Anhydrase B with Bromoacetate and Iodoacetamide

Hydrogen Ion Equilibria and the Chemical Modification of Lysine and Tyrosine Residues in Bovine Carbonic Anhydrase B

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Human Carbonic Anhydrase. Protein Conformation and Metal Ion Binding^*