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.