Understanding the Biochemistry and Physiology of the Gamma Carbonic Anhydrases in Methanosarcina thermophila
Open Access
- Author:
- Zimmerman, Sabrina Anastasia
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 07, 2007
- Committee Members:
- James Gregory Ferry, Committee Chair/Co-Chair
Sarah Ellen Ades, Committee Member
Squire J Booker, Committee Member
Joseph M Bollinger Jr., Committee Member
Ming Tien, Committee Member
Juliette Lecomte, Committee Member - Keywords:
- carbonic anhydrase
aceticlastic methanogensis
hydrogen bond network - Abstract:
- To date, there are five independently evolved classes (ƒÑ-, ƒÒ-, ƒ×-, ƒÔ-, and ƒê-) of carbonic anhydrases (CAs) participating in many physiological functions. Although the ƒ×-class is widely distributed, only one CA (Cam from a methanoarchaeon Methano-sarcina thermophila) has been characterized while no physiological function has yet been documented. The work presented in this thesis contributes to the general understanding of the biochemistry of CAs by kinetically investigating Cam, the prototypic ƒ×-class CA. In these studies, Cam was shown to utilize a hydrogen bond network analogously to the hydrogen bond network described for the ƒÑ- and ƒÒ-class CAs that involves Cam active site residues Gln75 and Asn73. In addition, Cam residue Asn202 may orient and polarize the incoming carbon dioxide molecule much like the backbone amide of ƒÑ-class residue Thr199. Therefore, the ƒÑ-, ƒÒ-, and ƒ×-class CA, Cam, each independently evolved variations of a fundamental functions that are essential for catalysis. This thesis also presents the initial characterization of the newly identified second ƒ×-class isozyme, CamH, thus significantly increasing the understanding of the ƒ×-class. CamH possesses several properties significantly different from Cam. Contrary to Cam, CamH does not possess a signal peptide, suggesting CamH is cytosolic whereas Cam is located at the membrane. Preliminary kinetic analyses reveal the rate of CO2 hydration in CamH (kcat/Km =104 M-1s-1) is two-fold lower than observed in Cam (kcat/Km =10 6 M-1s-1). Despite the kinetic differences, both CamH and Cam are up-regulated during aceticlastic methanogenesis. Therefore, a model was proposed based on this information outlining roles for Cam and CamH in acetate uptake in Methanosarcina species.