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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
NN
NN
NH2
O
PPPO
N
NH2
O
OH OHOHOH
NH
O
N
NH
R
NN
NN
NH2
O
PPPO
OH OHOHOH
NH
O
N
NR
N
NH2
O
NAD+ EF - His
+
-H+
+
EF-ADP ribose
+
Potential Mechanisms?
ON
NH2
O
OH OH
O
OH OH
O
OH OH
EF
ON
NH2
O
OH OH
O
OH OH
EF
N
NH2
O
NAD+
+ +EF (Nu:)
+EF (Nu:)
+
-OR-
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
Activates incoming nucleophile
3-point binding?
NH
O
N
NH
R
O
OHOH
N
NH2
O
+
Role of Glutamic Acid in the TS?
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!
H
O H
O
OHOH
N
NH2
O
+
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?
?*
Grow organismIsolate metabolite & look for marker
**
“feed” the labelled compound to the organism
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
O-Na+
O
O
OH
OH
?
For example:
• In the 1950’s, 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
O
OH
OH OH
CO2
6-MSA
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
O-Na+
O OH
H2O
O2
Examples:
OHO-Na+
O
• 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
CD3
O
OH
OH
D
DCD3 O
O
Proposed Pathway:
CD3
O
OH
OH
D
D
CD3 O
O
O
O
enzyme
CD3
ODD
D
D
O
O
O
CD3
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.,
Why?• (An in-depth look at these principles is beyond the scope
of this course)• Recall vibration model of a bond:
CH3OH CH3O- H+
CH3OD CH3O- D+
+
+
lightk
heavyk
r
Force constant, F
• 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 spectroscopye.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
• (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
C13 X
H
H H
C13
H
HH
Nu X+Nu: +
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
H
O H
O
OHOH
N
NH2
O
+