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

E. coli MEP synthase: steady-state kinetic analysis and substrate binding.

Andrew T Koppisch1, David T Fox, Brian S J Blagg

  • 1Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.

Biochemistry
|January 5, 2002
PubMed
Summary
This summary is machine-generated.

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

Attempts to Achieve Targeted Covalent Inhibition of Hsp90β.

Chemical biology & drug design·2026
Same author

Cell-Impermeable Hsp90α-Selective Inhibitor NDNA4 Inhibits Cancer Cell Migration and Targets Extracellular Hsp90α's Critical F-5 Fragment.

ACS chemical biology·2026
Same author

The evolution of heat shock protein 90 C-terminal inhibitors: From novobiocin to potential clinical candidates.

Cell stress & chaperones·2026
Same author

Optimization of triazole-based small molecule disruptors of the Aha1/Hsp90 complex that manifest potent activities.

European journal of medicinal chemistry·2026
Same author

Small Molecules to Elevate Rab7-GTPase Activity and Lower Cholesterol Accumulation in Niemann-Pick Type C Disease.

Pharmaceutical research·2026
Same author

Multi-omic integration identifies broad drug resistance mechanisms and strategies to therapeutically reprogram cancer cells.

iScience·2026

2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) catalyzes a key step in isoprenoid biosynthesis. Kinetic analysis reveals an ordered mechanism where NADPH binds before DXP, providing insights into MEP pathway regulation.

Area of Science:

  • Biochemistry
  • Enzymology
  • Metabolic Pathways

Background:

  • Isoprenoids are essential molecules derived from the methylerythritol phosphate (MEP) pathway.
  • 2-C-Methyl-D-erythritol-4-phosphate synthase (MEP synthase) is a crucial enzyme in this pathway.
  • Understanding MEP synthase kinetics is vital for metabolic engineering and drug development.

Purpose of the Study:

  • To characterize the steady-state kinetics and reaction mechanism of E. coli MEP synthase.
  • To elucidate the substrate binding order for MEP synthase.

Main Methods:

  • Purification of recombinant E. coli MEP synthase using ion-exchange and hydrophobic interaction chromatography.
  • Determination of steady-state kinetic constants (kcat, KM) and equilibrium constant (Keq).

Related Experiment Videos

  • Enzyme inhibition studies using fosmidomycin and dihydro-NADPH to probe the reaction mechanism.

    • Main Results:

    • MEP synthase exhibits kcat = 116 +/- 8 s(-1), K(M)(DXP) = 115 +/- 25 microM, and K(M)(NADPH) = 0.5 +/- 0.2 microM.
    • The reaction is reversible with K(eq) = 45 +/- 6.
    • Inhibition patterns indicate an ordered substrate binding mechanism, with NADPH binding prior to DXP.

    Conclusions:

    • MEP synthase follows an ordered kinetic mechanism where NADPH is the initial substrate.
    • These findings enhance our understanding of isoprenoid biosynthesis regulation.
    • The kinetic data can inform the design of inhibitors targeting the MEP pathway.