Carbonic anhydrase catalytic cycle

X-ray diffraction studies on several forms of the enzyme have demonstrated that the active site is composed of a pseudo-tetrahedral zinc center coordinated to three histidine imidazole groups and either a water molecule [(His)3Zn-OH2]2+ (His = histidine), or a hydroxide anion [(His)3Zn-OH] +, depending upon pH (156,157). On the basis of mechanistic studies, a number of details of the catalytic cycle for carbonic anhydrase have been elucidated, as summarized in Scheme 22... 

Scheme 22. Proposed catalytic cycle for carbonic anhydrase.

As an illustration, we briefly discuss the SCC-DFTB/MM simulations of carbonic anhydrase II (CAII), which is a zinc-enzyme that catalyzes the interconversion of CO2 and HCO [86], The rate-limiting step of the catalytic cycle is a proton transfer between a zinc-bound water/hydroxide and the neutral/protonated His64 residue close to the protein/solvent interface. Since this proton transfer spans at least 8-10 A depending on the orientation of the His 64 sidechain ( in vs. out , both observed in the X-ray study [87]), the transfer is believed to be mediated by the water molecules in the active site (see Figure 7-1). To carry out meaningful simulations for the proton transfer in CAII, therefore, it is crucial to be able to describe the water structure in the active site and the sidechain flexibility of His 64 in a satisfactory manner. 

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. 

Figure 9.5 Catalytic cycle for the hydration of C02 catalyzed by carbonic anhydrase II. Taken from http //chemlearn.chem.indiana.edu/C430/C430L16.pdf.

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... 

All the above structural and kinetic information obtained under a variety of conditions with different metal ions can be used to propose a catalytic cycle for carbonic anhydrase (Figure 2.21). As shown by studies on the pH-dependent properties of native and metal-substituted CAs, both type-I and type-II proteins have two acidic groups, the zinc-coordinated water and a free histidine. At... 

Fig. 28.22 (a) Schematic representation of the active site in human carbonic anhydrase II (CAII). (b) The catalytic cycle for the hydration of CO2 catalysed by CAII. 

Macrocyclic polyamine Zn +-complexes not only were used as carbonic anhydrase models. They were also shown to promote hydrolytic cleavage of phosphate esters and in particular phosphate diesters as present in DNA. In Figure 3, the catalytic cycle for monometallic activation is shown. Dissociation of a metal-bound water molecule provides a metal hydroxide species, which nucleophilically attacks the phosphorus center of a phosphate diester, whereby finally the aUcoxide gets released and the product is formed. 

Fig. 9.56 The mechanism of the hydration of COj by carbonic anhydrase. In the first two steps, a Lewis acid-base complex forms between the protein-boimd Zn ion and a water molecule, which is then deprotonated. In the next steps, COj binds to the active site and then reacts with the boimd OH ion, forming a bicarbonate ion. Release of the bicarbonate ion poises the enzyme for another catalytic cycle.

After many years of research, the first protein that uses Cd naturally has been discovered in marine diatoms a carbonic anhydrase with Cd as its catalytic center (CDCA) [9,10]. It appears that CDCA plays a pivotal role in the acquisition of inorganic carbon in diatoms, and thus the use of Cd in CDCA provides a link between the biogeochemical cycles of carbon and Cd. The existence of CDCA is an example of the unique mechanisms phytoplankton have evolved over geological times as an adaptation to life in the metal-poor environment of surface seawater. But CDCA may not be the only biological use of Cd in seawater. While we are beginning to understand how and how much Cd is utilized by phytoplankton cells, there are still many challenging questions that need to be answered. 

---