The zinc-requiring carbonic anhydrases (CA) are ubiquitous throughout the plant, animal, and bacterial kingdoms, and no other family of enzymes characterized by such catalytic, cellular, tissue, and health-related diversity as the seven known isozymes found in humans. Although the in vivo chemical function of the seven known human isozymes is to catalyze the hydration of carbon dioxide to yield bicarbonate plus a proton in the reaction CO2 + H2O ( HC03- + H+, this deceptively simple chemical function is required for diverse biological functions such as acid-base balance, CO2 transfer, ion exchange, respiration, fluid secretion, and biosynthesis. The seven isozymes exhibit similarities as well as contrasts in the chemical mechanism of CO2 hydration, and this proposal focuses on the structural chemistry of intramolecular proton transfer in isozymes II, IV, and V. Specifically, it is proposed to (1) determine the structures of CAII variants with zinc altered binding sites; (2) determine the structures of different CAV variants prepared to pinpoint the trajectory of catalytic proton transfer to buffer; (3) determine the structures of the soluble portion of membrane-anchored human and murine CAIVs, and to use these structures as a starting point for (a) analyzing structure-stability relationships among the CA isozymes, and (b) understanding structural aspects of diffusion-controlled catalysis conserved in the face of extraordinary evolutionary drift between CA isozymes II and IV. The proposed work will answer the question: what structural features constitute an effective catalytic proton shuttle site in an enzyme?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045614-08
Application #
6138432
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Flicker, Paula F
Project Start
1991-08-01
Project End
2001-12-31
Budget Start
2000-01-01
Budget End
2000-12-31
Support Year
8
Fiscal Year
2000
Total Cost
$174,334
Indirect Cost
Name
University of Pennsylvania
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104