113Cd NMR as a probe of the active sites of metalloenzymes

Armitage, Ian M.; Uiterkamp, A.J.M. Schoot; Chlebowski, J F; Coleman, Joseph E.

doi:10.1016/0022-2364(78)90160-9

Cited by 36 publications

(31 citation statements)

References 44 publications

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“…Our results deviate from those of Armitage et al (8,9) in two respects: we observed "13Cd NMR signals for hCAaseB at pH 8.9 and below (which they did not),* and we found no hCAaseB resonance as high upfield as 146 ppm. Earlier, we reported a hCAaseB resonance at pH 9.7 at 228 ppm.…”

Section: Resultscontrasting

confidence: 57%

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Cadmium-113 NMR of carbonic anhydrases: effect of pH, bicarbonate, and cyanide.

Jonsson¹,

Tibell²,

Evelhoch³

et al. 1980

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

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ABSTRACT"13Cd-Substituted human and bovine erythrocyte carbonic anhydrases have been studied by "13Cd NMR as a function of pH and bicarbonate concentration. Plots of chemical shift versus pH give sigmoidal titration curves in the pH range of the study, 6.9 to 10.5. The pKa values vary from 9.2 to 9.7, which correlates well with available activity profiles for the Cd-enzymes. Because the samples contain no buffers and no anions other than hydroxide, the results point to the existence of high and low pH forms of the enzymes in rapid exchange and differing in inner sphere coordination. When bicarbonate is added to the samples, upfield shifts are produced which eventually level off. Only a single CN-binds to the metal for all three enzymes. These observations are best explained by a rapid exchange among three species in which the open coordination site of the metal ion is occupied by hydroxide, water, or bicarbonate, as in the scheme: EOH-;± E-H20 ± E-HCQ3. Carbonic anhydrases (carbonate hydro-lyase, EC 4.2.1.1) are zinc metalloenzymes that are found in animals, plants, and certain bacteria and catalyze the reversible hydration of carbon dioxide (CO2 + H20 :=± HCO% + H+), the hydrolysis of certain esters, and various other reactions (1). Human carbonic anhydrase B (hCAaseB) and C (hCAaseC) and bovine carbonic anhydrase (bCAase) from human and bovine erythrocytes are monomeric enzymes of molecular weight k29,000, each molecule containing a single equivalent of firmly bound Zn(II) which is required for catalytic activity. The Zn can be replaced by various divalent metal cations, including Co(II) and Cd(II) (2). The activity appears to be controlled by a single titratable group with a pKa value around 7; the high pH form of the enzyme is required in the hydration reaction and the low pH form is required in the dehydration. A parallel between the spectral changes of Co(II)-enzymes and their activities versus pH has been taken as support for a mechanism in which the low and high pH forms are zinc-coordinated water and hydroxide ion, respectively (3, 4). However, the identity of the catalytic group remains in dispute, and alternative mechanisms have been proposed (5).In this report we present a systematic 1"3Cd NMR study of hCAaseB, hCAaseC, and bCAase as a function of pH and bicarbonate concentration. Such 113Cd NMR studies of metalloproteins have been shown to provide information regarding the coordination sphere of the metal ion (6-10).MATERIAL AND METHODS Enzymes. hCAaseB, hCAaseC, and bCAase were prepared by affinity chromatography according to the Khalifah et al. (11) modification of the method of Osborne and Tashian (12), followed by chromatography on DEAE-cellulose (Whatman DE-23). Enzyme concentrations were estimated spectrophotometrically by using the following extinction coefficients (Ag9o) and molecular weights: 18.7 cm-1, 16.3 cm-1, and 19.0 cm-1 and 29,300, 28,850, and 29,500 for hCAaseC, hCAaseB, and bCAase, respectively (1, 13,14). The metal-free enzymes were prepared by dialysis against 75 mM pyridine-2,4...

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Section: Resultscontrasting

confidence: 57%

“…Such 113Cd NMR studies of metalloproteins have been shown to provide information regarding the coordination sphere of the metal ion (6)(7)(8)(9)(10) …”

mentioning

confidence: 99%

Cadmium-113 NMR of carbonic anhydrases: effect of pH, bicarbonate, and cyanide.

Jonsson¹,

Tibell²,

Evelhoch³

et al. 1980

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

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“…oxide} Zn(II) is also a mimetic of carbonic anhydrase and is a weak catalyst in both the CO2-hydration and p-nitrophenyl acetate-hydrolysis assays (2). Similarly, model compounds that mimic carboxypeptidase and are capable of catalysis have been synthesized (14).…”

Section: Designed Sites With Three Protein Ligandsmentioning

confidence: 99%

The Design of Metal-Binding Sites in Proteins

Regan¹

1993

Annu. Rev. Biophys. Biomol. Struct.

157

114

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“…They can be a serious health hazard, not only to humans but to all living organisms, because of their ability to bind certain metalloenzymes and enzyme substrates containing thiol groups [9]. Cd(II) ions are chemically similar to Zn(II) and can replace them in apoproteins, inhibiting or distorting the catalytic activity of the metalloenzymes [10]. The best example of Zn(II) Cd(II) replacement occurs in metallothioneins (MT) [11].…”

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confidence: 99%