Carbonic Anhydrase: New Insights for an Ancient Enzyme

Abstract

proton shuttle residue 2- (N-morpholino) propanesulfonic acid Carbonic anhydrase catalyzes the reversible hydration of CO2 (Equation 1).CO2+H2O⇄HCO3−+H+Equation 1 The first carbonic anhydrase was purified from erythrocytes in 1933 (1Meldrum N.U. Roughton F.J. J. Physiol. (Lond.). 1933; 80: 113-142Crossref Scopus (333) Google Scholar) followed by the characterization of several mammalian isozymes that dominated research on carbonic anhydrase until recently. Although it has been known since the 1940s that carbonic anhydrase is ubiquitous in plants (2Bradfield J.R.G. Nature. 1947; 159: 467-468Crossref PubMed Scopus (20) Google Scholar), where it is essential for CO2 fixation, relatively few studies had been reported. Until 1994, only five carbonic anhydrases had been purified from prokaryotes; however, a recent survey has established that the enzyme is widely distributed among phylogenetically and physiologically diverse prokaryotes, indicating a far greater role for this enzyme in nature than previously recognized (3Smith K.S. Jakubzick C. Whittam T.S. Ferry J.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15184-15189Crossref PubMed Scopus (335) Google Scholar). The comparison of sequences and crystal structures of the mammalian and plant enzymes demonstrates that they evolved independently and have been designated the α- and β-class, respectively. An additional independently evolved γ-class was reported in 1994 (4Alber B.E. Ferry J.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6909-6913Crossref PubMed Scopus (203) Google Scholar) for which phylogenetic analyses predict an ancient origin (3Smith K.S. Jakubzick C. Whittam T.S. Ferry J.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15184-15189Crossref PubMed Scopus (335) Google Scholar). This review discusses dramatic advances over the past 3 years regarding the structure and biological chemistry of carbonic anhydrases. The three classes have no significant sequence identity, and the overall folds (Fig. 1) underscore their independent origins (5Liljas A. Laurberg M. EMBO J. 2000; 1: 16-17Crossref Scopus (49) Google Scholar). Despite gross structural differences, the active sites of all three classes function with a single zinc atom essential for catalysis (6Lindskog S. Pharmacol. Ther. 1997; 74: 1-20Crossref PubMed Scopus (774) Google Scholar, 7Christianson D.W. Cox J.D. Annu. Rev. Biochem. 1999; 68: 33-57Crossref PubMed Scopus (328) Google Scholar). Kinetic studies indicate that all three classes employ a two-step isomechanism (8Northrop D.B. Simpson F.B. Arch. Biochem. Biophys. 1998; 353: 288-292Crossref Scopus (23) Google Scholar). The first step is the nucleophilic attack of a zinc-bound hydroxide ion on CO2(Equation 2). The second step is the regeneration of the active site by ionization of the zinc-bound water molecule and removal of a proton from the active site (Equation 3). In this step, the zinc ion acts as a Lewis acid to lower the pKa of the water molecule from ∼14 to 7.0.Zn2+­OH−+CO2⇄Zn2++HCO3−Equation 2 Zn2++H2O⇄H++Zn2+­OH−Equation 3 Most carbonic anhydrases have kcat values greater than 104 s−1, which requires an intermediate PSR1 (Equation4) to transfer the proton from the metal-bound water molecule to the external buffer, “B” (Equation 5).PSR+Zn2+­H2O⇄Zn2+­OH−+PSR­H+Equation 4 PSR­H++B⇄PSR+B­H+Equation 5 Proton transport from the active site is the rate-limiting step for enzymes with kcat > 104s−1. Thus, kcat is a reflection of the rate of proton transport (Equation 3), whereas the catalytic efficiency (kcat/Km) is more reflective of the hydration step (Equation 2) and is insensitive to the rate of proton transport. The following sections focus on recent results revealing specific properties of the three classes of carbonic anhydrase, which provide new structural and biochemical perspectives for this enzyme. The α-class is the best characterized with 11 isozymes identified in mammals. Several isozymes are implicated in various disease states for which treatment frequently involves the application of sulfonamides that inhibit carbonic anhydrase activity. Earnhardt et al. (9Earnhardt J.N. Qian M. Tu C. Lakkis M.M. Bergenhem N.C. Laipis P.J. Tashian R.E. Silverman D.N. Biochemistry. 1998; 37: 10837-10845Crossref PubMed Scopus (43) Google Scholar) summarize sulfonamide inhibition constants and maximal kcat andkcat/Km values for CO2 hydration by isozymes I–VII. Prokaryotic α-class enzymes are few compared with the other two classes. The recent characterization of mammalian and prokaryotic α-class enzymes has been reported and reviewed (10Smith K.S. Ferry J.G. FEMS Microbiol. Rev. 2000; 24: 335-366Crossref PubMed Google Scholar, 11Chirica L.C. Elleby B. Lindskog S. Biochim. Biophys. Acta. 2001; 1544: 55-63Crossref PubMed Scopus (55) Google Scholar, 12Hewett-Emmett D. Tashian R.E. Mol. Phylogenet. Evol. 1996; 5: 50-77Crossref PubMed Scopus (526) Google Scholar, 13Huang S. Xue Y. Sauer-Eriksson E. Chirica L. Lindskog S. Jonsson B.H. J. Mol. Biol. 1998; 283: