Carbonic anhydrase II

General acid-base catalysis is often the controlling factor in many mechanisms and acts via highly efficient and sometimes intricate proton transfers. Whereas log K versus pH profiles for conventional acid-base catalyzed chemical processes pass through a minimum around pH 7.0, this pH value for enzyme reactions is often the maximum. In enzymes, the transition metal ion Zn2+ usually displays the classic role of a Lewis acid, however, metal-free examples such as lysozyme are known too. Good examples of acid-base catalysis are the mechanisms of carbonic anhydrase II and both heme- and vanadium-containing haloperoxidase. 

A simple calculation reveals that the picture cannot be quite as simple. Carbonic anhydrase has an exceptionally high overall rate of reaction, its turnover number kcat is -5 x 105 s-1 consequently, the rate constants of individual steps must be greater than this number. The acid dissociation of a Zn11 aqua species seems to be inconsistent with this requirement. The dissociation constant fQ can be written as the ratio of forward k and backward kh rate constants [Eq. (9.20)]. 

Therefore k = 10-7kb, and with kbmax = 1010 m-1 s-1 (the diffusional limit), Eq. (21) follows, showing an observed rate constant at least 1000-fold slower than expected. 

The mechanism can only be retained if a base very much better than water is present at the enzyme active site to deprotonate the Zn(OH2)2+ species. In fact, the structure determination reveals a histidine residue with its side chain positioned approximately halfway down the -15 A-deep cleft in the protein structure within which the Zn site is located. This arrangement could act as a proton shuttle between the Zn(OH2)2+ and external solvent water, possibly via another two water molecules also found within the cleft. As a consequence, the enhancement of ligand acidity by Zn11 is more important in the kinetic than the equilibrium sense (taken from http //www.chem.uwa.edu.aU/enrolled students/BIC sect4/sect4.2.htmll. 

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Aminoimidazole-4-Carboxamide Ribonucleotide Transformylase Carbonic Anhydrase II DNA Gyrase... 

Carbonic anhydrase II inhibitors, IC50 = 0.6 nM (left) and 0.8 nM (right)... 

Grtineberg S, Wendt B, Klebe G. Subnanomolar inhibitors from computer screening a model study using human carbonic anhydrase II. Angew Chem Int Ed Engl 2001 40 389-393 Angew Chem 2001 113 404-8. 

Figure 48-12. Schematic illustration of some aspects of the role of the osteoclast in bone resorption. Lysosomal enzymes and hydrogen ions are released into the confined microenvironment created by the attachment between bone matrix and the peripheral clear zone of the osteoclast. The acidification of this confined space facilitates the dissolution of calcium phosphate from bone and is the optimal pH for the activity of lysosomal hydrolases. Bone matrix is thus removed, and the products of bone resorption are taken up into the cytoplasm of the osteoclast, probably digested further, and transferred into capillaries. The chemical equation shown in the figure refers to the action of carbonic anhydrase II, described in the text. (Reproduced, with permission, from Jun-queira LC, Carneiro J BasicHistology. Text Atlas, 10th ed. McGraw-Hill, 2003.)...

Osteopetrosis (marble bone disease), characterized by increased bone density, is due to inability to resorb bone. One form occurs along with renal tubular acidosis and cerebral calcification. It is due to mutations in the gene (located on chromosome 8q22) encoding carbonic anhydrase II (CAII), one of four isozymes of carbonic anhydrase present in human tissues. The reaction catalyzed by carbonic anhydrase is shown below ... 

Sundaram, V., Rumbolo, P., Grubb, J., Strisciuglio, P, and Sly, W. S., Carbonic anhydrase II deficiency Diagnosis and carrier detection using differential enzyme inhibition and inactivation. Am. J. Hum. Genet. 38,125-136 (1986). 

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. 

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). 

Sly WS, Hewett-Emmett D, Whyte MP, Yu YS, Tashian RE (1983) Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc Natl Acad Sci USA 80 2752-2756... 

Figure 12. Oligoglycine-linked para-substituted benzenesulfonamides as inhibitors of carbonic anhydrase II.

A Jain, S. G. Huang, G. M. Whitesides, Lack of Effed of the Length of Oligoglydne- and 01igo(ethylene glycol)-Derived para-Substituents on the Affinity of Benzene-sulfonamides for Carbonic Anhydrase II in Solution ,/ Am. Chem. Soc. 1994,116,5057-5062. 

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. 

Fig. 14. Paramagnetic enhancements to water NMRD profiles for solutions of cobalt(II) human carbonic anhydrase I at pH 9.9 and 298 K ( ) (48,49) and for solutions of the nitrate adduct of cobalt(II) bovine carbonic anhydrase II at pH 6.0 and 298 K ( ) (126). The dashed line shows the best fit profile of the former data calculated with including the effect of ZFS, whereas the dotted line shows the best fit profile calculated without including the effect of ZFS.

Water NMRD profiles acquired for other complexes and proteins always exhibit the same features of hexaaqua nickel(II). As an example, we report here the profile of the hexa-coordinate nickel(II)-substituted bovine carbonic anhydrase II 54,55) (Fig. 15). As in the aqua complex, (i) the low-field profile is flat, (ii) no dispersion appears, the cOg dispersion being quenched in S = 1 complexes with large static ZFS 56) (see Section I.A.5) and the... 

Rowlett and Silverman used a Brpnsted plot to examine the interaction of external buffers with human carbonic anhydrase II. The buffers act as proton acceptors in the removal of the proton generated by the enzyme-catalyzed reaction. The Brpnsted plot displays a plateau at a value of about 10 for the catalytic rate... 

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