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Related Experiment Videos

Substrate recognition by the human fatty-acid synthase.

Loretha Carlisle-Moore1, Chris R Gordon, Carl A Machutta

  • 1Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, USA.

The Journal of Biological Chemistry
|October 11, 2005
PubMed
Summary
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Human fatty-acid synthase (HFAS) is a cancer drug target. Lysine-1699 is crucial for enoyl reductase (ER) activity and NADPH binding, and unexpectedly influences beta-ketoacyl reductase (BKR) activity, suggesting allosteric regulation.

Area of Science:

  • Biochemistry
  • Enzymology
  • Drug Discovery

Background:

  • Human fatty-acid synthase (HFAS) is a validated target for anti-cancer drug development.
  • Understanding the enoyl reductase (ER) active site is key for designing HFAS inhibitors.
  • Lysine-1699 (K1699) has been implicated in ER activity via pyridoxal 5'-phosphate modification.

Purpose of the Study:

  • To investigate the role of Lys-1699 in NADPH and substrate recognition by the ER active site.
  • To elucidate the contribution of Lys-1699 to the catalytic mechanism and stability of HFAS.
  • To explore the impact of K1699 mutations on both ER and beta-ketoacyl reductase (BKR) activities.

Main Methods:

  • Site-directed mutagenesis of Lys-1699 to Alanine (K1699A) and Glutamine (K1699Q).

Related Experiment Videos

  • Enzyme kinetics assays to determine kinetic parameters (kcat, Km) for HFAS reactions.
  • Thermodynamic analysis of NADPH binding to assess stabilization energies.

    • Main Results:

    • K1699A and K1699Q mutations significantly reduced kcat and kcat/KNADPH for the overall HFAS reaction (8- and 600-fold decreases, respectively).
    • Lys-1699 stabilizes the transition state by 4 kcal/mol and the ground state by 3 kcal/mol after NADPH binding.
    • Mutations at K1699 unexpectedly affected BKR activity, indicating a potential link between ER and BKR active sites.

    Conclusions:

    • Lys-1699 is critical for ER activity, directly involved in NADPH binding and catalysis.
    • The unexpected modulation of BKR activity by K1699 mutations suggests an allosteric mechanism connecting the ER and BKR NADPH binding sites.
    • These findings provide insights into HFAS structure-function relationships and potential allosteric drug targets.