Escherichia coli Cystathionine γ-Synthase Does Not Obey Ping-Pong Kinetics. Novel Continuous Assays for the Elimination and Substitution Reactions.

Cystathionine γ-synthase (CGS) is a pyridoxal phosphate-dependent enzyme that catalyzes a γ-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[sub catR]/ K[sub Mr, sup L-OSHS] = 9.8 × 10[sup 4] M[sup -1] s[sup -1], where the subscript R denotes the replacement reaction). The mechanism becomes ping-pong (k[sub catR]/g[sub Mr, sup L-OSHS] = 4.9 × 10[sup 4] M[sup -1] s[sup -1]) at L-Cys concentrations lower than K[sub m, sup L-Cys]. The enzyme also catalyzes a competing γ-elimination reaction, in which L-OSHS is hydrolyzed to succinate, NH[sub 3], and α-ketobutyrate (k[sub catE]/K[sub Me, sup L-OSHS] = 1350 ± 90 M[sup -1] s[sup -1], where the subscript E denotes the elimination reaction). The k[sub cat]/K[sub m, sup L-OSHS] versus Ph profile of ECGS is bell-shaped for both reactions. The Ph optimum and the Pk[sub 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 α-amino group of L-OSHS has a Pk[sub a] of 9.71 ± 0.01; therefore, neither limb of the k[sub cat]/K[sub m, sup L-OSHS] versus Ph profiles can be assigned to aldimine, or to L-OSHS prototropy. Novel continuous assays for the elimination reaction, employing D-2hydroxyisocaproate dehydrogenase, and for the substitution reaction, employing cystathionine β-lyase and L-lactate dehydrogenase as coupling enzymes, are described. [ABSTRACT FROM AUTHOR]