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Study of Enzyme Mechanisms

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Isotopes. Atoms of the same element having different numbers of neutrons ... exploit a unique property of isotope & detect using analytical techniques ... – PowerPoint PPT presentation

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Title: Study of Enzyme Mechanisms


1
Study of Enzyme Mechanisms
  • We have studied the mechanisms of peptide bond
    formation hydrolysis by an enzyme
  • Why study mechanisms?
  • Structure activity relationships ? understand
    protein folding, etc
  • Understand superfamilies
  • Design enzyme inhibitors
  • Correct a metabolic imbalance
  • Kill an organism Herbicides/pesticides,
    antibiotics

2
Diphtheria Toxin
Active peptide
  • Corynebacterium diphtheriae
  • ADP-ribosyltransferase
  • EF NAD ? ADP-EF nicotinamide
  • Mechanism also present in other toxins
  • Pertussis, E.Coli
  • Binding to EF (eukaryotes) blocks translation

3
Reaction
4
Potential Mechanisms?
-OR-
5
Active Site with NAD Bound (1st Step)
Hydrophobic interactions
Nu
6
Testing of Mechanism
  • Role of tyrosine?
  • Substitute with Phe ? small drop in catalytic
    activity
  • Substitute with Ala ? 105 drop in activity!
  • ? likely responsible for substrate recognition
    (hydrophobic interactions)
  • Other mutations show small effects
  • Key residues?
  • Glu-148 His-21
  • Mutations show large drop in catalytic activity
  • Glu148Ser 103 drop in activity

7
Plays a role in NAD binding
3-point binding?
Activates incoming nucleophile
8
Role of Glutamic Acid in the TS?
9
2 possible mechanisms?
  • In the absence of EF, hydrolysis of NAD will
    occur
  • Model the TS understand how stabilization of TS
    occurs
  • Occurs via an SN2 mechanism!

10
Diphtheria Toxin as a Drug?
  • Few successful inhibitors of the diphtheria toxin
    have been found
  • Instead, the toxins apoptotic inducing activity
    has been exploited to kill Cancer cells
  • Active site is maintained
  • Alter its targeting ability (to cell receptor)
  • Target toxin
  • Targeting polypeptide toxic peptide (DT)

Cell receptor
Cell death
11
Determination of Mechanism?
  • How do we elucidate a biological pathway or an
    enzymes mechanism?
  • Biological Methods genetic engineering
  • Construction of mutants
  • Chemical Methods
  • Construct analogues (recall the use of fluorine
    in tRNA)
  • Photochemical methods
  • Isotopes (stable radioactive)
  • OR can use a combination of both methods!

12
Isotopes
  • Atoms of the same element having different
    numbers of neutrons ? different masses
  • e.g. 1H, 2H, 3H 12C, 13C, 14C
  • Can be used as markers ? exploit a unique
    property of isotope detect using analytical
    techniques
  • Radioactivity
  • NMR activity
  • Markers can
  • Elucidate a biosynthetic pathway
  • Provide mechansitic (transition state) information

13
Early Days - Radioisotopes
14
Stable Isotopes
  • Commonly used 2H, 13C, 18O 15N
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