19th BCCE

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Program Management System - 19th BCCE - Teaching Bioanalytical Chemistry and Biochemistry Laboratory - Design, isolation and characterization of mutant cystathionine-beta lyase

Paper Title: Design, isolation and characterization of mutant cystathionine-beta lyase
Symposium: Teaching Bioanalytical Chemistry and Biochemistry Laboratory
Session: Session 1 of 1 [2006-07-31 09:00:00 - 12:00:00]
Paper Start Time: 10:35:00
Author: Greg Muth
Presenter: Greg Muth
CoAuthors: Joe Chihade
Abstract: The experimental biochemistry course at St. Olaf College provides an environment for students to participate in a hypothesis based laboratory study of a single enzymatic reaction using a variety of biochemical methods. We have chosen to use cystathionine b-lyase (CBL), a bacterial enzyme catalyzing the penultimate step in the pyridoxyl 5'-phosphate (PLP) dependent hydrolysis of L-cystathionine to L-homocysteine, pyruvate and ammonia. This enzyme was chosen 1) because the kinetic assays are robust, reproducible and can be monitored by visible spectroscopy 2) the enzyme utilizes several commercially available substrates allowing for comparison of specificity between different molecules and 3) there is a published crystal structure of the enzyme. Two goals of the course are to provide an environment for creativity and to encourage intellectual and physical continuity between experiments. To meet these goals, over the course of a thirteen-week semester up to eight junior and senior biochemistry students have studied the chemical mechanism of CBL through its organic reaction mechanism from substrate to product. This analysis paired with study of the crystal structure of the protein lead students to develop unique hypotheses regarding how a change in the sequence of the protein may lead to an altered function of the protein. The isolated proteins were analyzed physically by SDS-PAGE electrophoresis and functionally by kinetic assays. Rigorous calculation and presentation of the kinetics data allowed the students to compare the affects of their mutant as well as the other mutant proteins developed and analyzed by their classmates.
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