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