These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Escherichia coli cystathionine gamma-synthase does not obey ping-pong kinetics. Novel continuous assays for the elimination and substitution reactions.
    Author: Aitken SM, Kim DH, Kirsch JF.
    Journal: Biochemistry; 2003 Sep 30; 42(38):11297-306. PubMed ID: 14503880.
    Abstract:
    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.
    [Abstract] [Full Text] [Related] [New Search]