Kinetic and Spectroscopic Characterization of the Gamma-Carbonic Anhydrase from the Methanoarchaeon Methanosarcina thermophila ² Birgit E. Alber, ‡ Christopher M. Colangelo, § Jun Dong, § Christina M. V. Stålhandske, § Teaster T. Baird, | Chingkuang Tu, ⊥ Carol A. Fierke, | David N. Silverman, ⊥ Robert A. Scott, § and James G. Ferry* ,‡ Department of Biochemistry and Molecular Biology, The PennsylVania State UniVersity, UniVersity Park, PennsylVania, 16802, Center for Metalloenzyme Studies, UniVersity of Georgia, Athens, Georgia 30602, Department of Biochemistry, Duke UniVersity Medical Center, Durham, North Carolina, 27710, and Department of Pharmacology and Therapeutics, UniVersity of Florida, College of Medicine, GainesVille, Florida, 32610 ReceiVed December 8, 1998; ReVised Manuscript ReceiVed July 21, 1999 ABSTRACT: The zinc and cobalt forms of the prototypic γ-carbonic anhydrase from Methanosarcina thermophila were characterized by extended X-ray absorption fine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and 18 O exchange equilibrium assays. EXAFS results indicate that cobalt isomorphously replaces zinc and that the metals coordinate three histidines and two or three water molecules. The efficiency of either Zn-Cam or Co-Cam for CO 2 hydration (k cat /K m ) was severalfold greater than HCO 3 - dehydration at physiological pH values, a result consistent with the proposed physiological function for Cam during growth on acetate. For both Zn- and Co-Cam, the steady-state parameter k cat for CO 2 hydration was pH-dependent with a pK a of 6.5-6.8, whereas k cat / K m was dependent on two ionizations with pK a values of 6.7-6.9 and 8.2-8.4. The 18 O exchange assay also identified two ionizable groups in the pH profile of k cat /K m with apparent pK a values of 6.0 and 8.1. The steady-state parameter k cat (CO 2 hydration) is buffer-dependent in a saturable manner at pH 8.2, and the kinetic analysis suggested a ping-pong mechanism in which buffer is the second substrate. The calculated rate constant for intermolecular proton transfer is 3 × 10 7 M -1 s -1 . At saturating buffer concentrations and pH 8.5, k cat is 2.6-fold higher in H 2 O than in D 2 O, suggesting that an intramolecular proton transfer step is at least partially rate-determining. At high pH (pH > 8), k cat /K m is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. Therefore, at high pH the catalytic mechanism of Cam appears to resemble that of human CAII, despite significant structural differences in the active sites of these two unrelated enzymes. Carbonic anhydrases catalyze the reversible hydration of carbon dioxide (eq 1) and are ubiquitous in all three phylogenetic domains of life: the Archaea, Eucarya, and Bacteria. On the basis of sequence comparisons, there are three distinct classes (R, , and γ) that appear not to share a common ancestor (1-3). All three classes contain a catalytic zinc ion that has been structurally characterized by EXAFS 1 for both the R (4) and  (5, 6) classes, but not the γ class. Although a crystal structure has not been reported for the  class, EXAFS indicates that the zinc atom is coordinated to one histidine, two cysteines, and one to two solvent molecules. Crystal structures have been determined for five isozymes (I-V) of the monomeric human carbonic anhydrase belong- ing to the R class (7-11). The crystal structure has also been determined for the trimeric Cam from the methanoarchaeon Methanosarcina thermophila (12). Cam is the prototype of the γ class and the only procaryotic carbonic anhydrase for which a structure is known. The drastic departure in the tertiary and quaternary structure of Cam compared to the human isozymes provides further evidence that the R- and γ-classes did not evolve from a common ancestor, yet the ² This work was supported by grants from the National Institutes of Health to C. A. F. (GM40602), D. N. S. (GM25154), R. A. S. (GM42025), and J. G. F. (GM44661). The XAS data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL), which is operated by the Department of Energy, Division of Chemical Sciences. The SSRL Biotechnology program is supported by the National Institutes of Health, Biomedical Resource Technology Program, Divi- sion of Research Resources. Support for the X-ray fluorescence detector is from NIH BRS Shared Instrumentation Grant RR05648. CMC was partially supported by the NSF Research Training Group Award to the Center for Metalloenzyme Studies (DIR 90-14281). * To whom correspondence should be addressed. Telephone: 814- 863-5721. Fax: 814/863-6217. E-mail:jgf3@psu.edu. ‡ Department of Biochemistry and Molecular Biology, The Penn- sylvania State University. § Center for Metalloenzyme Studies, University of Georgia. | Department of Biochemistry, Duke University Medical Center. ⊥ Department of Pharmacology and Therapeutics, University of Florida. 1 Abbreviations: EXAFS, extended X-ray absorption fine structure; Cam, Methanosarcina thermophila carbonic anhydrase produced in Escherichia coli; apo-Cam, metal-depleted Cam; Zn-Cam, zinc- reconstituted apo-Cam; Co-Cam, cobalt-reconstituted apo-Cam; MES, 2-(N-morpholino) ethanesulfonic acid; TAPS, N-tris(hydroxymethyl)- methyl-3-aminopropanesulfonic acid; MOPS, 2-(N-morpholino)pro- panesulfonic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane- sulfonic acid; XAS X-ray absorption spectroscopy. CO 2 + H 2 O a HCO 3 - + H + (1) 13119 Biochemistry 1999, 38, 13119-13128 10.1021/bi9828876 CCC: $18.00 © 1999 American Chemical Society Published on Web 09/17/1999