Fermentchemische Charakterisierung von Carbonat-Hydrolasen (Carboanhydrasen) aus menschlichen Erythrocyten

Abstract

Carbonic anhydrase, a Zn-containing enzyme, catalyzes the hydration of CO₂ and the dehydration of H₂CO₃. These properties make it the powerful promoter of the CO₂ uptake in the tissue and the discharge in the lungs. Besides this function, carbonic anhydrase is also of importance in other biological processes. The enzyme is widely distributed in the animal kingdom, in plants, and it has also been found in certain bacteria.
The most abundant source of human carbonic anhydrase is the red cell. The enzyme is isolated and purified by chromatography of erythrocyte hemolyzate on DEAE-Sephadex columns. Other methods employ zone electrophoresis or column chromatography on Amberlite IRC-50 of chloroform-ethanol treated hemolyzate. By these methods, the enzyme is obtained in three different, distinct, active fractions, designated A, B and C. The most plentiful component is enzyme B, whereas the A- and C- forms are present only in low concentrations. The three components differ considerably in some of their properties. Physicochemical parameters are compiled in Table 1 in the text. The carbonic anhydrase molecule has a highly compact structure and the molecular shape is close to spherical.
The Zn atom can be dissociated reversibly by dialysis against o-phenanthroline. The dissociation rate at pH 5.0 in the presence of the chelating agent is twice as fast as at pH 7.0. The metalfree apoenzyme is enzymatically inactive; activity is restored by adding Zn ions. Enzymatic activity is directly proportional to the amount of protein-bound Zn.
The Zn atom can be replaced by other bivalent heavy-metal ions, such as Co⁺⁺, Ni⁺⁺, Fe⁺⁺, Mn⁺⁺, Cd⁺⁺, Cu⁺⁺ and Hg⁺⁺. The Co derivative is about half as active as compared to the native Zn enzyme. The other metalloproteins possess but a low degree of residual activity or are entirely inactive, as for the Cu and Hg derivatives. The Co enzyme shows four absorption maxima between 510 and 650 mp. Spectral shifts of these maxima induced by various carbonic anhydrase inhibitors, and the direct relation between enzyme activity and Zn content have led to the conclusion that the Zn atom is the essential constituent of the active center.
The sulfhydryl group of the sole cysteine residue in carbonic anhydrase is not free to titrate in the native enzyme. It is accessible only in the apoenzyme or after the Zn has been split off by denaturation, either by acid or urea treatment. Yet, the SH group apparently does not serve as a ligand group of the Zn atom, since crystallographic studies showed that the distance between the Zn and S atom is close to 15 Å.
Carbonic anhydrase shows catalytic versatility: it also catalyzes the reversible hydration of aldehydes (acetaldehyde) and accelerates the cleavage of esters (p-nitrophenyl acetate). These processes proceed at a considerably slower rate as compared to the reactions with CO₂ and H₂CO₃ substrates. The active center appears to be identical for all these reactions. Acetazolamide, a recognized sulfonamide inhibitor of carbonic anhydrase, efficiently inhibits aldehyde and esterase activities as well. However, esterase activity is not affected by diisopropylfluorophosphate.
Carbonic anhydrase is an inherent constituent of the red cell. So far, only two cases are known with a markedly reduced level of this enzyme; complete absence of erythrocyte carbonic anhydrase in man has not been observed. The enzyme forms – above all enzyme B – show considerable genetic variability. Of the human B form, four different variants were found, differing in electrophoretic mobility and esterase activity. Enzyme C seems to be evolutionally older than enzyme B; the latter may have evolved by duplication and further changes of the carbonic anhydrase C locus.