Carbonic anhydrase catalytic mechanism

Supuran, C.T. Carbonic anhydrases catalytic and inhibition mechanism, distribution and physiological roles. In Carbonic Anhydrase Its Inhibitors and Activators, ed. Supuran, C.T., Scozzafava, A., Conway, J., CRC Press Boca Raton, FL, 2004, pp. 1-24. 

The first zinc enzyme to be discovered was carbonic anhydrase in 1940, followed by car-boxypeptidase A some 14 years later. They both represent the archetype of mono-zinc enzymes, with a central catalytically active Zn2+ atom bound to three protein ligands, and the fourth site occupied by a water molecule. Yet, despite the overall similarity of catalytic zinc sites with regard to their common tetrahedral [(XYZ)Zn2+-OH2] structure, these mononuclear zinc enzymes catalyse a wide variety of reactions, as pointed out above. The mechanism of action of the majority of zinc enzymes centres around the zinc-bound water molecule,... 

C. K. Tu, D. N. Silverman, C. Forsman, B. H. Jonsson, S. Lindskog, Role of Histidine 64 in the Catalytic Mechanism of Human Carbonic Anhydrase II Studied with a Site-Specific Mutant , Biochemistry 1989, 28, 7913-7918. 

The results of site-directed mutagenesis analysis of zinc ligands of higher plant p-carbonic anhydrase and of P. purpureum carbonic anhydrase have confirmed that zinc is essential for catalysis. X-ray fine structure data indicated that a water molecule is hydrogen bonded to the zinc-ligated Asp-151 and Asp-405. The water molecule is not directly coordinated to the zinc atom. A possible catalytic mechanism of C02 hydration cycle (211) has been proposed as given in Scheme 10. 

Roberts et al. reported a 27 kDa monomeric carbonic anhydrase, TWCAl, from the marine diatom Thalassiosira weissflogii (221). X-ray absorption spectroscopy indicated that the catalytic zinc is coordinated by three histidines and a water molecule, similar to the active sites of the a- and y-CAs (222). Also, the active site geometry is similar to that of a-CAs. Based on these results the catalytic mechanism is expected to be similar to that of the -class carbonic anhydrases. Tripp et al. (223) proposed that this TWCAl is the prototype of a fourth class carbonic anhydrase designated as 8-class CAs. In the... 

The calculated [using a quantized classical path (QCP) approach] and observed isotope effects and rate constants are in good agreement for the proton-transfer step in the catalytic reaction of carbonic anhydrase. This approach takes account of the role of quantum mechanical nuclear motions in enzyme reactions.208... 

Before discussing the wider concepts of the active site and the catalytic mechanism of carbonic anhydrase, it will be considered to what extent the bonding of Co(II) can be predicted on the basis of the spectral evi-... 

Until more concrete structural information is obtained, the discussion on the catalytic mechanism of carbonic anhydrase must remain rather speculative. The experimental evidence requires the presence in the active site of a basic group being in some manner linked to the metal ion. This group is generally thought to play a critical role either as a nucleophile in a direct reaction with the substrate, or through general base catlysis. Several schemes for the function of carbonic anhydrase have been proposed (16, 41, 50, 78, 79) ... 

Some of the current ideas of the catalytic site of carbonic anhydrase are summarized in Fig. 12. As discussed in Section III A 2, little is known about the binding of C02. The possibility of a transient metal-coordinated CO2 (16) is open. No effort has been made to sketch the details of the catalytic mechanism as Fig. 12 undoubtedly will have to be revised after the completion of the crystal structure determination. 

The catalytic mechanism for C02 hydration-dehydration by carbonic anhydrase represents the focal issue of the present discussion. We have to consider two aspects (1) the mode of binding of the C02 substrate at the active site, and (2) the physical-chemical state of ligands on the zinc ion. 

Carbonic anhydrase II, present in human red blood cells (RBCs), catalyzes the reversible hydration of C02. It is one of the most efficient enzymes and only diffusion-limited in its turnover numbers. The catalytic Zn11 is ligated by three histidine residues and OH this ZnOH+ structure renders the zinc center an efficient nucleophile which is able to attack the C02 molecule and capture it in an adjacent hydrophobic pocket. The catalytic mechanism is shown in Figure 9.5. 

S. Lindskog and J.E. Coleman. 1973. The catalytic mechanism of carbonic anhydrase Proc. Natl. Acad. Sci. USA 70 2505-2508. (PubMed)... 

B. H. Jonsson, H. Steiner, and S. Lindskog. 1976. Participation of buffer in the catalytic mechanism of carbonic anhydrase PEBBPetr. 64 310-314. (PubMed)... 

The catalytic mechanism of carbonic anhydrase has been investigated by Pullman (1981), Cook et al. (1984), Liang and Lipscomb (1988), Merz et al. (1989) and Jacob et al. (1990), who studied gas-phase models using molecular orbital methods. The special importance of electrostatics in the action of this enzyme has been stressed by several authors in the last two decades (Sawaryn and Sokalski, 1979 Sheridan and Allen, 1981 Ressler, 1982 Krauss and Garmer, 1991 Aqvist and Warshel, 1992, 1993). 

Figure 13. A simplified model for the catalytic mechanism for human carbonic anhydrase (a), invariant residues near the catalytic zinc ion (b).

It has been suggested that in certain parts of the oceans where dissolved zinc levels are extremely low, the limited availability of Zn might also limit CO2 fixation. This is because the Mg -dependent enzyme which fixes CO2, RuBisCO (ribulose-l,5-bisphosphate carboxylase/oxygenase), is a notoriously inefficient enzyme, with a low affinity for its substrate, CO2. It therefore requires high concentrations of CO2, and while cyanobacteria have evolved extremely effective mechanisms for uptake of CO2, which is then converted to HCOj, they need to regenerate CO2 from HCOj" in the vicinity of RuBisCO, and this requires the Zn -dependent enzyme, carbonic anhydrase. This was the first Zn -dependent enzyme to be discovered (Chapter 12) and virtually all carbonic anhydrases discovered to date have a catalytic Zn ion, bound to one His and two Cys residues. 

The mechanism of action of mononuclear zinc enzymes depends on the Zn +-OH2 centre, which can participate in the catalytic cycle in three distinct ways (Figure 12.2) — either by ionisation, to give zinc-bound hydroxyl ion (in carbonic anhydrase), polarisation by a general base (in carboxypeptidase), or displacement of... 

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