Cystathionine gamma-synthase (CGS) is a pyridoxal phosphate-dependent enzyme that catalyzes a gamma-replacement reaction, in which the succinyl group of an O-succinyl-L-homoserine (L-OSHS) is displaced by the thiol of L-cysteine to form L-cystathionine, in the first step of the bacterial transsulfuration pathway. The mechanism of Escherichia coli CGS (eCGS) is ordered with L-OSHS associating before L-Cys (k(catR)/K(mR)(L-OSHS) = 9.8 x 10(4) M(-1) s(-1), where the subscript R denotes the replacement reaction). The mechanism becomes ping-pong (k(catR)/K(mR)(L-OSHS) = 4.9 x 10(4) M(-1) s(-1)) at L-Cys concentrations lower than K(m)(L-Cys). The enzyme also catalyzes a competing gamma-elimination reaction, in which L-OSHS is hydrolyzed to succinate, NH(3), and alpha-ketobutyrate (k(catE)/K(mE)(L-OSHS) = 1350 +/- 90 M(-1) s(-1), where the subscript E denotes the elimination reaction). The k(cat)/K(m)(L-OSHS) versus pH profile of eCGS is bell-shaped for both reactions. The pH optimum and the pK(a) values for the acidic and basic limbs are 7.4, 6.8 +/- 0.1, and 8.0 +/- 0.1, respectively, for the elimination reaction and 7.8, 7.4 +/- 0.1, and 8.3 +/- 0.1, respectively, for the replacement reaction. The internal aldimine of eCGS remains protonated at pH <10.5, and the alpha-amino group of L-OSHS has a pK(a) of 9.71 +/- 0.01; therefore, neither limb of the k(cat)/K(m)(L-OSHS) versus pH profiles can be assigned to aldimine, or to L-OSHS prototropy. Novel continuous assays for the elimination reaction, employing D-2-hydroxyisocaproate dehydrogenase, and for the substitution reaction, employing cystathionine beta-lyase and L-lactate dehydrogenase as coupling enzymes, are described.