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Journal of Bacteriology, December 2000, p. 6605-6613, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Structural and Kinetic Characterization of an Archaeal beta -Class Carbonic Anhydrase

Kerry S. Smith,1 Nathaniel J. Cosper,2 Christina Stalhandske,2,dagger Robert A. Scott,2 and James G. Ferry1,*

Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802,1 and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602-25562

Received 17 May 2000/Accepted 11 September 2000

The beta -class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the Spinacia oleracea (spinach) beta -class carbonic anhydrase indicate that the secondary structure of the beta -class enzymes is predominantly alpha -helical, unlike that of the alpha - or gamma -class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters kcat and kcat/Km for CO2 hydration are pH dependent. The steady-state parameter kcat is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, kcat is 2.1-fold higher in H2O than in D2O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter kcat/Km is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic beta -class carbonic anhydrases have fundamental characteristics similar to the eukaryotic beta -class enzymes and firmly establish that the alpha -, beta -, and gamma -classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802. Phone: (814) 863-5721. Fax: (814) 862-6217. E-mail: jgf3{at}psu.edu.

dagger Present address: Sandvikvagen, S-35241 Vaxjo, Sweden.

Journal of Bacteriology, December 2000, p. 6605-6613, Vol. 182, No. 23
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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