Date of Award

2015

Document Type

Thesis

Degree Name

Master of Science

School

College of Arts and Sciences

Program

Biology MS

First Advisor

Peter J. Lyons

Second Advisor

Robert Zdor

Third Advisor

Marlene Murray

Abstract

Metallocarboxypeptidases are found in most organisms and function in the digestion and maturation of proteins. Ecm14 is a putative metallocarboxypeptidase found in the yeast Saccharomyces cerevisiae vacuole. The function of Ecm14 as an enzyme has been unclear due to the presence of active site amino acids not typically found in metallocarboxypeptidases, suggesting either no enzymatic activity or a unique mechanism. In order to investigate the enzymatic mechanism of Ecm14, expression of histidine-tagged Ecm14 protein was attempted in human HEK293T cell culture, S. cerevisiae, and baculovirus expression systems. No expression was detected in HEK293T cells in preliminary experiments. Expression in the yeast system resulted in insolubility of Ecm14, regardless of induction time, temperature, or inducer concentration.

In contrast, following expression of Ecm14 in Sf9 cells using the baculovirus system, approximately 31% of Ecm14 was soluble and detected as a 40 kDa histidine-tagged protein by western blotting. Sf9-expressed Ecm14 was purified using metal affinity chromatography. No enzymatic activity could be detected for purified Ecm14 in the presence of substrate consisting of chromogenic 3-(2-furyl) acryloyl conjugated to a C-terminal dipeptide, PhePhe. Activation of Ecm14 by enzymatic removal of the prodomain was successful with the addition of chymotrypsin. Ecm14 cleaved of its prodomain showed activity in the presence of the above substrate with a preference for substrate at a pH of 6.0, similar to the known pH of the yeast vacuole. Ecm14 showed activity towards substrate proportional to the amount of enzyme present, suggesting that Ecm14 is non-catalytic in activity. Ecm14 can bind to substrate and limit the activity of a carboxypeptidase with known activity, CPA1, indicating that Ecm14 binds and cleaves but does not release the cleavage products.

Subject Area

Sacchromyces cerevisiae, Carboxypeptidases

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