Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Equilibrium perturbation by isotope substitution.

M I Schimerlik, J E Rife, W W Cleland

    Biochemistry
    |December 2, 1975
    PubMed
    Summary

    Enzyme reactions involving deuterium-labeled malate reveal slower C-D bond cleavage than C-H bonds, impacting hydride transfer and equilibrium. This sensitive method quantifies isotope effects, aiding in understanding enzymatic mechanisms.

    Related Concept Videos

    You might also read

    Related Articles

    Articles linked to this work by shared authors, journal, and citation graph.

    Sort by
    Same author

    pH-rate profiles support a general base mechanism for galactokinase (Lactococcus lactis).

    FEBS letters·2013
    Same author

    Oxamate is an alternative substrate for pyruvate carboxylase from Rhizobium etli.

    Biochemistry·2013
    Same author

    Catalytic mechanism of perosamine N-acetyltransferase revealed by high-resolution X-ray crystallographic studies and kinetic analyses.

    Biochemistry·2012
    Same author

    A kinetic and isotope effect investigation of the urease-catalyzed hydrolysis of hydroxyurea.

    Biochemistry·2010
    Same author

    13C isotope effect on the reaction catalyzed by prephenate dehydratase.

    Biochimica et biophysica acta·2009
    Same author

    A heavy-atom isotope effect and kinetic investigation of the hydrolysis of semicarbazide by urease from jack bean (Canavalia ensiformis).

    Biochemistry·2008

    Area of Science:

    • Biochemistry
    • Enzymology
    • Isotope Effects

    Background:

    • Enzymatic reactions involving malate are crucial in metabolic pathways.
    • Understanding kinetic isotope effects provides insights into enzyme mechanisms.
    • Deuterium labeling is a common technique for studying reaction pathways.

    Purpose of the Study:

    • To investigate the kinetic isotope effects of malic enzyme and malate dehydrogenase using deuterium-labeled malate.
    • To develop a sensitive method for determining isotope effects and equilibrium constants.
    • To explore the impact of deuterium substitution on enzyme reaction kinetics and thermodynamics.

    Main Methods:

    • Enzyme assays using malate-2-deuterium (malate-2-d) and nicotinamide adenine dinucleotide phosphate (TPNH).
    • Monitoring changes in TPNH levels to detect equilibrium perturbations.
    • Determination of kinetic isotope effects (KIEs) on V/Kmalate and V, and equilibrium constants (Keq).

    Main Results:

    • Slower cleavage of C-D bonds compared to C-H bonds was observed during hydride transfer.
    • Kinetic isotope effects for malic enzyme and malate dehydrogenase were determined to be 1.45 and 1.70-2.16, respectively.
    • Equilibrium constants were found to be lower for malate-2-d than for malate-2-h, with a factor of 0.76-0.82.

    Conclusions:

    • The observed isotope effects are attributed to differential C-D and C-H bond cleavage rates.
    • The developed method demonstrates high sensitivity for determining isotope effects, including for 13C and 15N.
    • Deuterium substitution significantly affects equilibrium constants, consistent with theoretical predictions.

    Related Experiment Videos