Date of Award

1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Sue G. Bartlett

Second Advisor

Harold Silverman

Abstract

It is the goal of this dissertation research to reveal some aspects of the physical nature of spinach carbonic anhydrase as a representative $\beta$CA using the techniques of sequence comparison, molecular biology, and biophysics. Though both $\alpha$ and $\beta$ carbonic anhydrases are zinc dependent metalloenzymes, it is clear that the two isoforms do not adopt the same mechanism for coordinating the active site metal. While $\alpha$CA binds zinc through three histidine ligands, $\beta$CA cannot due to a lack of evolutionarily conserved histidines. Instead, the $\beta$ family has adopted a ligand scheme incorporating a single histidine and two cysteines. This has been determined by systematically mutating possible zinc ligands in the spinach enzyme and then assaying the resulting variants for stoichiometric metal binding. Additionally, this conclusion is corroborated by inspection of the wild type enzyme's extended X-ray spectrum. This analysis indicates the metal is surrounded by two sulfur atoms and two nitrogen or oxygen species. Secondly, it has been long established that not only do the $\beta$ isoforms differ from their $\alpha$ cousins in their multimeric assembly, but subtypes exist within the $\beta$ family in which monocot forms assemble into lower molecular weight oligomers while dicot forms assemble into higher order structures. In an attempt to gain insight into the differences between monocot and dicot CAs, the CA cDNA from barley, a monocot, was sequenced. Analysis of the open reading frame revealed that the barley enzyme lacked ten amino acids at the carboxyl terminus which are conserved in the dicot isozymes. It is here demonstrated that this extension contributes to the difference in multimeric organization between monocots and dicots. When this extension is deleted from the spinach enzyme, the resulting mutant displays an apparent deficit in its ability to form higher order multimers. Furthermore, this carboxyl extension will interact with the CA holoenzyme in the yeast two-hybrid system showing that the observed characteristics of the deletion mutant do not arise from secondary disruptions, but rather the carboxyl terminus does participate in intermolecular interactions.

ISBN

9780591905137

Pages

66

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