Carbonic anhydrase stability

CO3 species was formed and the X-ray structure solved. It is thought that the carbonate species forms on reaction with water, which was problematic in the selected strategy, as water was produced in the formation of the dialkyl carbonates. Other problems included compound solubility and the stability of the monoalkyl carbonate complex. Van Eldik and co-workers also carried out a detailed kinetic study of the hydration of carbon dioxide and the dehydration of bicarbonate both in the presence and absence of the zinc complex of 1,5,9-triazacyclododecane (12[ane]N3). The zinc hydroxo form is shown to catalyze the hydration reaction and only the aquo complex catalyzes the dehydration of bicarbonate. Kinetic data including second order rate constants were discussed in reference to other model systems and the enzyme carbonic anhy-drase.459 The zinc complex of the tetraamine 1,4,7,10-tetraazacyclododecane (cyclen) was also studied as a catalyst for these reactions in aqueous solution and comparison of activity suggests formation of a bidentate bicarbonate intermediate inhibits the catalytic activity. Van Eldik concludes that a unidentate bicarbonate intermediate is most likely to the active species in the enzyme carbonic anhydrase.460... 

J.D. Badjic and N.M. Kostic, Effects of encapsulation in sol-gel silica glass on esterase activity, conformational stability, and unfolding of bovine carbonic anhydrase II. Chem. Mater. 11, 3671-3679 (1999). 

Given the reaction and the very high stability constants involved, the production of cobalt carbonic anhydrase would require a solution not of ACS-grade cobalt nitrate but a 99.999999999999. .. 999% pure cobalt nitrate solution. What happened in the lab synthesis was that trace metals in the ACS-grade salt were selectively bound to the apo-carbonic anhydrase because their stability constant advantage was orders of magnitude over that of cobalt. The sample used to discover this was sub-milligram in mass. 

Subsequent to CO2 association in the hydrophobic pocket, the chemistry of turnover requires the intimate participation of zinc. The role of zinc is to promote a water molecule as a potent nucleophile, and this is a role which the zinc of carbonic anhydrase II shares with the metal ion of the zinc proteases (discussed in the next section). In fact, the zinc of carbonic anhydrase II promotes the ionization of its bound water so that the active enzyme is in the zinc-hydroxide form (Coleman, 1967 Lindskog and Coleman, 1973 Silverman and Lindskog, 1988). Studies of small-molecule complexes yield effective models of the carbonic anhydrase active site which are catalytically active in zinc-hydroxide forms (Woolley, 1975). In addition to its role in promoting a nucleophilic water molecule, the zinc of carbonic anhydrase II is a classical electrophilic catalyst that is, it stabilizes the developing negative charge of the transition state and product bicarbonate anion. This role does not require the inner-sphere interaction of zinc with the substrate C=0 in a precatalytic complex. 

The engineering of zinc-binding sites in a-helical peptides, where metal binding stabilizes protein tertiary structure, has been reported by Handel and DeGrado (1990). In these experiments zinc-binding sites are incorporated into a dimeric helix-loop—helix peptide (H3 2) and a protein composed of four helices connected by three short loop sequences (H3 4). a model of one subunit of the H3 2 dimer is found in Fig. 47. In addition to metal complexation by two histidine residues at positions n and n+4 of one a helix, the metal is coordinated by a third histidine residue of an adjacent a helix. The composition of the zinc coordination polyhedron is like that of carbonic anhydrase (i.e., Hiss), and spectroscopic results suggest that all three histidine residues are involved in zinc complexation. This work sets an important foundation... 

Examples of other recombinant enzymes in which an alteration using site-directed mutagenesis resulted in altered substrate binding efficiencies, rates of catalysis, or stability include carbonic anhydrase (Alexander, Nair Christianson, 1991), lactate dehydrogenase (Feeney, Clarke Holbrook, 1990), and several industrially important proteases (Wells etal., 1987 Siezenera/., 1991 Teplyakovcra/., 1992 Aehle et al., 1993 Rheinnecker et al., 1994). 

One must marvel at the way various factors work in concert so that hemoglobin can be useful in multiple roles oxygen deliverer, carbon dioxide remover, and pH stabilizer. From the explanation we have given it should be clear why the hemoglobin is confined to cells in the blood rather than being present as a free plasma protein. The intracellular carbonic anhydrase and GBP of the erythrocytes are essential for efficient hemoglobin function. 

Table 4. Stabilities of bovine carbonic anhydrase — anion complexes...

Table 7. Apparent stability constants for metal binding in procarboxypeptidase A, carboxypeptidase A and human carbonic anhydrase B...

Quon et al. (1985) investigated the stability of esmolol in blood, plasma, red blood cells, and purified enzymes (human serum pseudocholinesterase, human and dog serum albumin, acetyl choline esterase, carbonic anhydrases A and B and human haemoglobin). Udata et al. (1999) studied the hydrolysis of propranolol ester prodrugs in purified acetylcholine esterase. 

Carbonic anhydrase has one Zn2+ ion per molecule of enzyme, and the metal ion resides in the active site. Aspartate carbamoyltransferase has six Zn2+ ions per dodecamer these are required fo the stabilization of the complex, since without Zn2+, the hexamer dissociates. 

---