CHARACTERIZATION OF THE ROLE OF THE LARGE AND SMALL SUBUNITS AND THE DISULFIDE BONDS ON CARBONIC ANHYDRASE’S ACTIVITY

Alexa R. Deemer, H. David Husic Department of Chemistry Lafayette College 730 High St, Easton, PA 18042

Carbonic anhydrase (CA), an enzyme important to the efficient utilization of carbon dioxide in photosynthetic organisms, catalyzes the reversible hydration of carbon dioxide to bicarbonate and protons. In the unicellular green alga, Chlamydomonas reinhardtii, the predominant form of CA is localized in the periplasmic space. The periplasmic CAs in C. reinhardtii are heterotetramers, consisting of two disulfide-bonded large subunits (36 kDa) and two small subunits (4 kDa), each connected to a large subunit by a disulfide bond. A goal in this research is to characterize the role of the large and small subunits and the disulfide bonds on CA’s catalytic activity. Ethylene glycol bis(succinimidyl succinate) (EGS), a bifunctional chemical cross-linking agent that contains an amino-reactive NHS-ester linked to a 12-atom spacer arm, was used to covalently crosslink the subunits of CA. Following treatment with EGS, dithiothreitol (DTT) was used to reduce the disulfide linkages between the subunits of CA, and the effect of DTT on the catalytic activity of the control or EGS-treated CA was determined. An electrophoretic analysis in the presence or absence of 2-mercaptoethanol confirmed the affect of EGS cross-linking of the subunits of CA. In the absence of EGS cross-linking, CA lost all catalytic activity when treated with DTT. When first cross-linked with EGS, CA retained almost full catalytic activity after subsequent DTT treatment. These results suggest that both the small and large subunits of CA are required for catalytic activity of the enzyme, but disulfide bonds between subunits and intra-chain disulfide bonds in the large subunit are not essential for catalysis.


Additional Abstract Information

Presenter: Alexa Deemer

Institution: Lafayette College

Type: Poster

Subject: Biochemistry

Status: Approved


Time and Location

Session: Poster 8
Date/Time: Fri 2:40pm-3:40pm
Location: University Center Ballroom - Tripod 5 Side B