Download 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
N
P
O
O
N
O
N
+
+
R
HN
N
H
OH
OH
OH
OH
NAD+
EF - His
-H+
NH2
N
N
N
N
O
P
P
O
O
N
H
N
OH
O
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?
OO
O
O
NH
NH
2
++
N
N
2
O
HO
HO
O
HO
HO
N
N
R
R
O
O
Glu148
N
N
HH
N
HN
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
• Markers can:
– Elucidate a biosynthetic pathway
– Provide mechansitic (transition state) information
*
*
Early Days - Radioisotopes
Stable Isotopes
• Commonly used: 2H, 13C, 18O & 15N