Carbonic anhydrases evolution

D.W. Christianson and C.A. Fierke (1996) Accounts of Chemical Research, vol. 29, p. 331 - Carbonic anhydrase Evolution of the zinc binding site by Nature and by design . 

Redox catalysis Zn, Fe, Cu, Mn, Mo, Co, V Se, Cd, Nl Enzymes (see Table 11.4 for more Information) Reactions with oxygen (Fe, Cu) Oxygen evolution (Mn) Nitrogen fixation (Fe, Mo) Inhibition of llpid peroxidation (Se) Carbonic anhydrase (Cd) Reduction of nucleotides (Co) Reactions with H2 (Nl) Bromoperoxidase activity (V)... 

We can use the two hypothetical steps of section Clb i.e., that kcJKM be maximized and that KM be greater than [S], to set up criteria for judging the state of evolution of an enzyme whose function is to maximize rate. We recall from Chapter 3 that the maximum value of kcJKM is the rate constant for the diffusion-controlled encounter of the enzyme and substrate, and from Chapter 4 that this is about 108 to 109 s "1 M l. A perfectly evolved enzyme should have a kcJKM in the range of 108 to 109 s"1 and a KM greater than [S]. Using the data for kcJKM listed in Table 4.4 and the substrate concentrations and KM values mentioned in this chapter, it appears that carbonic anhydrase and triosephosphate isomerase are perfectly evolved for the maximization of rate, which agrees with the conclusions of W. J. Albery and J. R. Knowles on triosephosphate isomerase.5... 

Less than 10 years after the discovery of carbonic anhydrase in 1932, this enzyme was found to contain bound zinc, associated with catalytic activity. This discovery, remarkable at the time, made carbonic anhydrase the first known zinc-containing enzyme. At present, hundreds of enzymes are knovm to contain zinc. In fact, more than one-third of all enzymes either contain bound metal ions or require the addition of such ions for activity. The chemical reactivity of metal ions—associated with their positive charges, with their ability to form relatively strong yet kinetically labile bonds, and, in some cases, with their capacity to be stable in more than one oxidation state—explains why catalytic strategies that employ metal ions have been adopted throughout evolution. 

The molecular components of many buffers are too large to reach the active site of carbonic anhydrase. Carbonic anhydrase II has evolved a proton shuttle to allow buffer components to participate in the reaction from solution. The primary component of this shuttle is histidine 64. This residue transfers protons from the zinc-bound water molecule to the protein surface and then to the buffer (Figure 9.30). Thus, catalytic function has been enhanced through the evolution of an apparatus for controlling proton transfer from and to the active site. Because protons participate in many biochemical reactions, the manipulation of the proton inventory within active sites is crucial to the function of many enzymes and explains the prominence of acid-base catalysis. 

A third family, the -carbonic anhydrases, also has been identified, initially in the archaeon Methanosarcina thermophila. The crystal structure of this enzyme reveals three zinc sites extremely similar to those in the a-carbonic anhydrases. In this case, however, the three zinc sites lie at the interfaces between the three subunits of a trimeric enzyme (Figure 9.31). The very striking left-handed P-helix (a P strand twisted into a left-handed helix) structure present in this enzyme has also been found in enzymes that catalyze reactions unrelated to those of carbonic anhydrase. Thus, convergent evolution has generated carbonic anhydrases that rely on coordinated zinc ions at least three times. In each case, the catalytic activity appears to be associated with zinc-bound water molecules. 

Convergent Evolution Has Generated Zinc-Based Active Sites in Different Carbonic Anhydrases... 

Convergent evolution has yielded three different classes of carbonic anhydrases. Mammals contain a-CA, plants and certain bacterial contain p-CA, while archea contain y-CA. The three classes of carbonic anhydrases differ in amino acid sequence and in their protein fold. Nevertheless, the active sites all contain a zinc ion (held by three histidine residues in the a- and y-CA, and by one histidine and two cysteines in the P-CA) and a hydrogen bond donor near the zinc and presumably catalyze the reaction in a similar manner. 

The other structural classes of carbonic anhydrases may also serve as protein ligands. The active site of the 3-CA is the approximate mirror image of that for a-CA. Nature s substrates, carbon dioxide and biocarbonate, are achiral so this mirror-image relationship is only an accident of convergent evolution. However, for enantioselective reactions, these two enzymes may form an enantiocomplementary pair and may catalyze reactions with opposite enantioselectivity. 

Using the carbonic anhydrases as examples, describe why convergent evolution is thought to have selected a common active-site structure. 

The data in Table 1 show that 18-7F, like wild type, induces the CO2 concentrating mechanism. Both external carbonic anhydrase activity and apparent photosynthetic affinity for CO2 increase dramatically in air-adapted cells. In spite of induction of the concentrating mechanism, 18-7F shows both oxygen and light inhibition of photosynthetic O2 evolution (Fig 1). 

Quantitatively carbon dioxide is the most important waste product. It is eliminated by the lungs. The establishment of the equilibrium between dissolved and gaseous carbon dioxide is catalyzed by carbonic anhydrase, a Zn-containing enzyme. Furthermore, hemoglobin becomes a stronger acid by addition of oxygen, i.e. it releases H+ ions, and thus enhances the evolution of CO2 from HCOa ... 

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