Site Specific Mutagenesis of H Pylori Carbonic Anhydrase

Rowlett, Roger

Abstract

Helicobacter pylori, a human gastric pathogen, codes for two different carbonic anhydrases in its genome, both an alpha and beta form. Because of the importance of carbonic anhydrase in processing cellular carbon dioxide, bicarbonate and protons--all products of the enzyme urease which H. pylori requires for survival in gastric mucosa--it is important to fully understand the structure and function of beta-carbonic anhydrases. One broad goal of the proposed research is to clone, overexpress, and functionally characterize beta-carbonic anhydrase from H. pylori and a closely related carbonic anhydrase from Arabidopsis thaliana. The enzymes will be functionally characterized by stopped-flow spectrophotometry and 13C-NMR exchange kinetics in order to identify the rate-determining step(s) in the mechanism of action, and to partially dissect the mechanism by measuring certain non-rate-determining steps. The enzymes will be structurally characterized by ICP-AES, electrophoresis, and submitted for X-ray crystallographic structure determination. A second broad goal of the proposed research is to further understand the catalytic mechanism and inhibitor binding characteristics of these beta-carbonic anhydrases by engineering site-directed mutants of beta-carbonic anhydrase using recombinant DNA techniques. Mutant enzymes will be overexpressed and functionally and structurally characterized in a manner similar to the wild-type enzymes. Especially interesting mutant enzymes will be submitted for X-ray crystallographic structure determination.