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Study of Enzyme Mechanisms

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We have studied the mechanisms of peptide bond formation & hydrolysis by an enzyme ... In the absence of EF, hydrolysis of NAD will occur ... – 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
  • Different mass (mass spec.)
  • Markers can
  • Elucidate a biosynthetic pathway
  • Provide mechansitic (transition state) information

13
How?
?

feed the labelled compound to the organism
Grow organism
Isolate metabolite look for marker


14
Early Days - Radioisotopes
  • Common markers
  • 14C (t1/2 5700 y, Nat. Abund. trace)
  • 3H (t1/2 12 y, Nat. Abund. 0)
  • 32P (t1/2 14 d, Nat. Abund 0)
  • Once metabolite is isolated, radioactivity
    (decay) is detected
  • Problems
  • Where is the isotope (marker)?
  • Harsh degradation methods must be employed ? can
    take weeks
  • Safety
  • Availability of precursor

15
For example
  • In the 1950s, Birch administered sodium acetate
    that was carbon-14 labelled to a Penicillium
    organism
  • Using harsh degradation methods, he was able to
    establish how sodium acetate was used to
    synthesize 6-MSA

16
Stable Isotopes
  • With the development of pulsed NMR came the use
    of stable isotopes ? gain information on
    connectivity
  • Mass spectrometry can be used ? little
    information location of isotope
  • Commonly used 2H, 13C, 18O 15N
  • Carbon-13
  • NMR active (I ½)
  • Nat. abundance 1.1
  • Many compounds are commercially available
  • Used to study the fate of carbon (i.e., C-C bonds
    formed bonds broken)

17
  • Deuterium
  • NMR active (I 1)
  • Nat. Abundance 0.015
  • Commercially available (i.e. D2O) cheap!
  • For the study of the fate or source of hydrogen
  • E.g. Which proton is deprotonated? Is a given
    proton from H2O or another molecule?
  • 18O and 15N
  • Employed to study the fate of oxygen and nitrogen
  • i.e., amino acids Did oxygen come from water or
    oxygen?

18
Precursors (what to feed)
  • Choice of isotope compound to feed depend on
    pathway
  • i.e.,
  • Sodium acetate is an intermediate in many
    biochemical pathways
  • Some knowledge of the pathway helps, but it is
    not necessary ? use knowledge of other pathways
  • Examples

19
Examples
20
  • Other possibilities
  • Neighboring carbon-13 labels ? 13C-13C (coupled
    doublets)
  • No change in signal intensity ? label did not
    incorporate into metabolite!
  • Deuterium?
  • Can use 2H NMR
  • Dont need to worry about background deuterium
    ? any deuterium signal seen, must come from your
    precursor

21
A Look Back at 6-MSA
Proposed Pathway
22
Mechanisms
  • Isotopes (stable and/or radio) can also be
    employed elucidate a mechanism (transition state)
  • Bases on the principle that there is a change in
    reactivity due to isotopic substitution
  • How?
  • Kinetic isotope effects (KIEs)
  • Can probe transition states directly ? useful for
    understanding cataylic processes
  • KIE lightk / heavyk

23
  • i.e.,
  • Why?
  • (An in-depth look at these principles is beyond
    the scope of this course)
  • Recall vibration model of a bond

Force constant, F
r
24
  • Total energy is proportional to the frequency of
    vibration
  • Related to force constant (unique to spring)
  • Related to mass
  • ? change mass, change frequency
  • Recall, we use the same model for IR spectroscopy
  • e.g. C-H (stretch) 2700 3300 cm-1
  • C-D (stretch) 2000 2400 cm-1
  • ? changing the mass can affect the rate at which
    a bond is cleaved or formed ? reaction rate!

CHCl3 CDCl3
25
  • (Can also use quantum mechanical methods)
  • Primary KIE
  • The effect is occurring on atom undergoing
    substitution
  • i.e.
  • ? A KIE can provide info on the change in the
    environment (vibrational) in going from reactant
    to TS

26
Measuring Isotope Effects
  • Competitive KIEs
  • Measure the rate constants
  • Labelled unlabelled reactants are combined in a
    single reaction mixture ? allowed to react as
    competitive substrates (enzymatic or
    non-enzymatic)
  • Measure the light/heavy at different times
  • End up with a KIEobs
  • If KIE 0, then no isotope effect ? atom is not
    near reacting site

27
  • How?
  • Measure isotopic ratios using
  • Mass spectrometry
  • Radioactivity (very efficient)
  • 3H, 14C 32P
  • Can also use NMR (not trivial!)

28
Application
  • Recall the hydrolysis of NAD
  • TS determined by KIEs
  • Isotopes used to determine that both Nu and
    nicotinamide ring are both in the TS
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