AbstractCystathionine β‐lyase (CBL) catalyzes the hydrolysis of L‐cystathionine (L‐Cth) to produce L‐homocysteine, pyruvate, and ammonia. A series of active‐site mutants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of residues R58, R59, D116, W340, and R372 in catalysis and inhibition by aminoethoxyvinylglycine (AVG). The effects of these mutations on the kcat/K for the β‐elimination reaction range from a reduction of only 3‐fold for D116A and D116N to 6 orders of magnitude for the R372L and R372A mutants. The order of importance of these residues for the hydrolysis of L‐Cth is: R372 >> R58 > W340 ≈ R59 > D116. Comparison of the kinetic parameters for L‐Cth hydrolysis with those for inhibition of eCBL by AVG demonstrates that residue R58 tethers the distal carboxylate group of the substrate and confirms that residues W340 and R372 interact with the α‐carboxylate moiety. The increase in the pKa of the acidic limb and decrease in the pKa of the basic limb of the kcat/K versus pH profiles of the R58K and R58A mutants, respectively, support a role for this residue in modulating the pKa of an active‐site residue.