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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

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.

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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.