Über die Reaktion von Carbonat-Hydro-Lyase mit p-Nitrophenylacetat

Schneider, F; Lieflaender, M

doi:10.1515/bchm2.1963.334.1.279

Cited by 17 publications

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“…the reversible reaction CO2 + H20 i: HCO3-+ H+ (1), the hydration of certain aldehydes (2), and the hydrolysis of certain esters (3,4). The enzyme occurs in plants (5,6), in certain bacteria (7), and in animals (8).…”

mentioning

confidence: 99%

Crystal structure of human erythrocyte carbonic anhydrase B. Three-dimensional structure at a nominal 2.2-A resolution.

Kannan¹,

Notstrand²,

Fridborg³

et al. 1975

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

242

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The three- Two major forms of carbonic anhydrase occur in human erythrocytes (10): the low activity form carbonic anhydrase B, and the high activity form carbonic anhydrase C. Carbonic anhydrase B is found in greater quantity than carbonic anhydrase C. The complete amino-acid sequences of the two isozymes have been established (11)(12)(13)(14) and show a great deal of homology. The three-dimensional structure of carbonic anhydrase C has been established to 2-A resolution (15, 16); the present article presents the high-resolution structure determination of carbonic anhydrase B. MATERIALS AND METHODSCrystallization and Heavy Atom Derivatives. Human carbonic anhydrase B was isolated from erythrocytes (kindly provided by the University Hospital, Uppsala) by mild methods (17). The enzyme was further purified by electrophoresis and on a Sulfur-ethylsephadex C-50 ion exchange column. Crystals of the native enzyme were prepared from 2.3 M ammonium sulfate, pH 8.7, by a seeding technique in small, thick-walled glass capillaries as described (18). Heavy atom derivatives were prepared by soaking native enzyme crystals for at least a week in 2.3 M ammonium sulfate solution, pH 8.7, containing 1 mM heavy atom salts, K2Au(CN)2, * This is paper II in a series. In all 30 layers, zero and upper levels were collected to cover about 85% of the reflections within the 2-A sphere. Intensity measurements were performed by a fully automatic microdensitometer constructed by Ing. V. Klimecki of the Department of Chemistry, University of Uppsala, in collaboration with our group. The program system used for data processing was developed by one. of us (K.K.K.) and is similar to that described by Jarup et al. (21).All the symmetry-related reflections were averaged, and the average amplitudes were used in subsequent calculations. The reflections in the 10-A sphere and reflections below a threshold were omitted.. Out of the 14,000 unique reflections thus measured, about 10,500 reflections were used in the refinements of heavy atom parameters, phase angle calculations, and electron density maps.Heavy Atom Parameters and Phase Angle Calculation. The heavy atom positional parameters were initially determined from difference Patterson and difference Fourier calculations in two orthogonal projections. A single site mercury derivative (18) was used in the two centrosymmetric projections to calculate the signs used in the difference Fouriers from which the principal heavy atom positions were determined for the other derivatives. Subsequent cycles of least-squares refinement and difference Fouriers included all the derivatives. This procedure did not introduce any serious biasing effect due to the heavy atom derivatives concerned. Threedimensional difference Patterson and correlation functions (22) were calculated to verify the correct assignment of the heavy atom positions. Alternate cycles of phase angle calculation and least-squares refinement were performed for relative scale factor, overall isotropic temperature factor, positional paramet...

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mentioning

confidence: 99%

Crystal structure of human erythrocyte carbonic anhydrase B. Three-dimensional structure at a nominal 2.2-A resolution.

Kannan¹,

Notstrand²,

Fridborg³

et al. 1975

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

242

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“…Thus, carbonic anhydrase catalyses the hydrolysis of 476 KINETICS OF CARBONIC ANHYDRASES certain esters (Schneider & Lieflander, 1963;Tashian, Douglas & Yu, 1964), and the hydration of certain aldehydes (Pocker & Meany, 1965) and even the hydrolysis of 2, 4-dinitrofluorobenzene (Edsall, 1969). Experimental investigation of these other catalytic functions as well as of the transport of volatile fatty acids and of ammonium must ultimately provide clues to the physiological role of the enzyme in the intestinal tract, a role which is reflected in the characteristic distribution of the enzyme.…”

Section: Possible Physiological Functions Of Isoenzymes In Intestinalmentioning

confidence: 99%

The isoenzymes of carbonic anhydrase: kinetic properties with particular reference to the functions in the intestinal tract

Carter¹,

Parsons²

1972

The Journal of Physiology

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1. Details are given of an electrometric method for measuring the activity of isoenzymes of carbonic anhydrase (EC 4.2.1.1) in catalysing the hydration of carbon dioxide under different conditions at 0° C. In the method, a measured volume of water saturated with carbon dioxide at a known partial pressure and appropriate temperature is introduced into a buffered solution. Using a sensitive electrometer and recording instrument, the subsequent change in hydrogen ion concentration is recorded as a function of time. Under the conditions of assay, the pH change induced in the presence of substrate is very small (ΔpH < 0·05 units) and the period of observation need not exceed 10 sec. 2. For enzymes isolated from guinea‐pig tissues, it is found that the specific activity of the ‘high activity’ isoenzyme (carbonic anhydrase C, carbonic anhydrase II, HACA) is about eighteen times that of the ‘low activity’ counterpart (carbonic anhydrase B, carbonic anhydrase I, LACA) when measured at 0° C, pH 7·2, and ionic strength 0·19. Under the same conditions, the Km was found to be 10 m M for the ‘high activity’ isoenzyme and 23 m M for the ‘low activity’ isoenzyme. No differences were found between the equivalent kinetic parameters of the corresponding isoenzymes isolated from different tissues. 3. The isoenzymes isolated from guinea‐pig tissues are found to be inhibited by acetazolamide in a non‐competitive manner. It is also found that the ‘high activity’ isoenzyme is many times more sensitive to this inhibitor than is the ‘low activity’ isoenzyme. Evidence is presented which indicates that one acetazolamide binding site is present on each molecule of either isoenzyme. 4. While chloride ions specifically inhibit the ‘low activity’ component of guinea‐pig carbonic anhydrase (I0·5 = 40 m M), acetate, butyrate and pyruvate inhibit both isoenzymes. Under the conditions employed, acetate and pyruvate are more strongly inhibitory to the ‘low activity’ isoenzyme than to the ‘high activity’ isoenzyme, while butyrate is more strongly inhibitory to the ‘high activity’ isoenzyme. 5. The findings are discussed with particular reference to the physiological significance of the presence of the isoenzymes in the gastro‐intestinal tract. Also considered are possible relationships between the distribution of the ‘low activity’ isoenzyme in these tissues and the transport and metabolism of products of fermentation occurring in the intestinal lumen.

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Biochemical Genetics of Carbonic Anhydrase

Tashian

Carter

1976

Advances in Human Genetics

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Über die Reaktion von Carbonat-Hydro-Lyase mit p-Nitrophenylacetat

Cited by 17 publications

References 0 publications

Crystal structure of human erythrocyte carbonic anhydrase B. Three-dimensional structure at a nominal 2.2-A resolution.

Crystal structure of human erythrocyte carbonic anhydrase B. Three-dimensional structure at a nominal 2.2-A resolution.

The isoenzymes of carbonic anhydrase: kinetic properties with particular reference to the functions in the intestinal tract

Biochemical Genetics of Carbonic Anhydrase

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