Exploration of Active-Site Residues of Escherichia Coli Cystathionine β-Lyase by Site-Directed Mutagenesis
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Cystathionine β-lyase is the second enzyme of the bacterial transsulfuration pathway, which catalyzes the reaction of L-cystathionine to pyruvate, ammonia, and L-homocysteine. CBL is dependant on the cofactor pyridoxal-5'-phosphate (PLP), which forms an internal aldimine with the catalytic lysine (eCBL-K210) in its inactive state. This work focuses on residues having a direct impact on the positioning of PLP and/or K210. Steady-state kinetics data from five site-directed D185 variants suggests that eCBL may be the only one of fold type I enzymes that performs the β-elimination reaction using E1 mechanism. The change in positioning of PLP caused by the substitutions of A207 and T209 causes enzymatic instability and inactivity, which does not occur with the Y211F variant. The R262K variant shows positive cooperation; a result that was not observed in our previously constructed eCBL variants. Finally, the importance of maintaining the aromatic properties of W340 was highlighted through its substitution.
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Copyright © 2018 the author(s). Theses may be used for non-commercial research, educational, or related academic purposes only. Such uses include personal study, research, scholarship, and teaching. Theses may only be shared by linking to Carleton University Institutional Repository and no part may be used without proper attribution to the author. No part may be used for commercial purposes directly or indirectly via a for-profit platform; no adaptation or derivative works are permitted without consent from the copyright owner.
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