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

摘要

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 andthe 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.