Transcript Study of Enzyme Mechanisms

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
Diphtheria Toxin
• Corynebacterium
diphtheriae
• ADP-ribosyltransferase
• EF + NAD+ → ADP-EF +
nicotinamide
• Mechanism also present
in other toxins
– Pertussis, E.Coli
• Binding to EF
(eukaryotes) blocks
translation
Active peptide
Reaction
O
NH2
NH2
N
N
N
P P P
N
O
O
N
O
N
+
+
R
HN
N
H
OH
OH
OH
OH
NAD+
EF - His
-H+
NH2
N
N
N
N
O
O
P P P
O
O
N
H
N
OH
OH
OH
EF-ADP ribose
OH
R
N
NH2
+
N
Potential Mechanisms?
O
NH2
+
O
+
N
EF (Nu:)
O
O
EF
OH
-OR-
OH
OH
OH
OH
OH
NAD+
O
NH2
+
O
N
EF (Nu:)
O
O
EF
OH
OH
OH
OH
NH2
+
N
Active Site with NAD+ Bound (1st Step)
Hydrophobic
interactions
Nu:
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
Plays a role in
NAD+ binding
3-point binding?
Activates incoming
nucleophile
Role of Glutamic Acid in the TS?
O
NH2
+
O
N
O
HO
HO
N
R
N
H
N
H
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!
O
NH2
+
N
O
HO
HO
O H
H
Diphtheria Toxin as a Drug?
• Few successful inhibitors of the diphtheria toxin have
been found
• Instead, the toxin’s apoptotic inducing activity has been
exploited to kill Cancer cells
– Active site is maintained
– Alter it’s targeting ability (to cell receptor)
– “Target toxin”
• Targeting polypeptide + toxic peptide (DT)
Cell receptor
Cell death
Determination of Mechanism?
• How do we elucidate a biological pathway or an
enzyme’s 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!
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
How?
?
*
“feed” the
labelled
compound
to the
organism
Grow organism
Isolate metabolite
& look for marker
*
*
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
For example:
• In the 1950’s, Birch administered sodium acetate that
was carbon-14 labelled to a Penicillium organism:
O
OH
O-Na+
OH
OH
OH
O
?
O
CO2
OH
6-MSA
• Using harsh degradation methods, he was able to
establish how sodium acetate was used to synthesize 6MSA
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)
• 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?
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
H2O
O
OH
O-Na+
O2
Examples:
O
O-Na+
OH
• 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
– Don’t need to worry about “background” deuterium  any
deuterium signal seen, must come from your precursor
A Look Back at 6-MSA
D
O
CD3
CD3
O
OH
D
OH
O
Proposed Pathway:
D
O
CD3
CD3
O
OH
D
OH
O
O
CD3
D D
O
O
O
O
CD3
enzyme
D
D
O
O
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
i.e.,
lightk
CH3OH
heavyk
CH3OD
+
CH3O-
+
H
CH3O-
+
D
+
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
• 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
CHCl3
CDCl3
•  changing the mass can affect the rate at which a bond
is cleaved or formed → reaction rate!
• (Can also use quantum mechanical methods)
• Primary KIE
– The effect is occurring on atom undergoing substitution
i.e.
H
Nu:
+
13
H
H
C X
H
Nu
13
+
C
H
X
H
 A KIE can provide info on the change in the environment
(vibrational) in going from reactant to TS
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
• How?
• Measure isotopic ratios using:
– Mass spectrometry
– Radioactivity (very efficient)
• 3H, 14C & 32P
– Can also use NMR (not trivial!)
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
O
NH2
+
N
O
HO
HO
O H
H