Transcript Three-Point Binding Model

Three-Point Binding Model
• First proposed by Ogsten (1948) to explain biological
enantioselection/enantiospecificity
• Serves as a model for chromatographic chiral stationary
phases
Preferential binding
occurs via intramolecular
non-covalent forces:
H-bonding
salt bridge
Ionic
Dipole-dipole
Van der Waals
Enantioselection by an Enzyme
HO
*
glycerol kinase
OH
OH
achiral
CH2OH moieties are
different because of
non-equivalent
binding sites in the
enzyme
2-
HO
H
OPO3
OH
3-glycerol phosphate
Three-Point Binding Model - Enantiospecificity
• Only one enantiomer binds to enzyme & is involved in
reaction
A
A
3 interactions
D
B
C
b
c
good fit, high H
d
D
B
b
C
c
d
With the other enantiomer…
A
C
1 interaction
D
poor fit, low H
B
b
d
c
B
A
b
2 interactions
D
poor fit, low H
C
c
d
•  we get enantiospecificity (substrate & biomolecule are
chiral)
• To do this efficiently, we need a large biomolecule to
align three binding sites to give high specificity
• One problem with model:
– Model is a static representation → “lock & key”
Binding
• The cost of binding:
E
+
S
K
E.S
(Michaelis Complex)
Km (Michaelis constant): small value indicates high affinity
for substrate
 Kbinding ( ~ 1/Km)
Strong binding → K > 1
ΔG= -RT ln K
ΔG must be –ve
ΔGbinding = ΔHbinding- TΔSbinding
For 2 molecules in, 1 out: ΔS is –ve
 (-TΔS) term is +ve
 Entropy disfavors binding of substrate to enzyme
 To get good binding, need –ve ΔH (i.e. bond formation)
• Each non-covalent interaction is small (H-bond ~ 5
kcal/mol), but still gives a –ve ΔH
• Enzymes use many FG’s to sum up many weak noncovalent interactions (i.e. 3 points)
Back to tyrosyl-tRNA synthase:
O
O
+
O
NH3
Adenosine
P O P O P O
O
O
O
O
O
+
O
O
NH3
O
P
Adenosine
O
OH
OH
R
O
R
O
+
B
O
NH3
O
OH
OH
tRNA Tyr
OH
B
OH
Tyrosyl-tRNA synthase
• Use binding to orient CO2- nucleophile adjacent to P
specifically as electrophile → specificity
• Many non-covalent interactions overcome entropy of binding:
H-bonds
Enz.Tyr .ATP
Tyrosine + ATP + Enz
Enz.Tyr--AMP + H2P2O73(released)
bind tRNA
OH
Enz.Tyr--OtRNA
+
AMP
Enz.Tyr--AMP . tRNA
(released)
Enz + Tyr--OtRNA
Can isolate this complex in
the absence of tRNA
Tyrosyl-tRNA Synthase.tyr-adenylate
Bind ATP
Binding AAs
*
3 point binding
enantiospecificity
ATP, not
dATP
Tyr specificity
* Main chain contacts
*
*
Orient  PO4 towards
CO2Increase P+
*
O
P
P
* Main chain contacts
*
• We have examined the crystal structure of tyrosyl-tRNA
synthase (Tyr & ATP bound)
– Key contacts
– 3 point binding model for (S)-tyrosine
• We inferred geometry of bound ATP prior to reaction (i.e.
ATP is no longer bound to enzyme)
Step 1:
• CO2- attacks PO42- () giving pentacoordinate P (trigonal
bipyramidal) intermediate
O
O
O
O
+
-O
P
-O
O
Ad
O
O
-O
P
O
-O
Ad
Step 2:
• Diphosphate must leave
• Cannot “see” this step  PPi has already left the
enzyme site in the crystal structure
• However, can use model building to include P & P of
ATP:

Thr40 & His45 form
H-bonds to P

**Stronger Hbonds are formed
in TS than in trig.
Bipyramidal
intermediate
Lower TS energy 
accelerate collapse of
intermediate

Gln195
Tests of Mechanism
1)
Site-directed mutagenesis
–
Replace Gln195 with Gly  (Gln195Gly)
• Rate slows by > 1000 fold
• ΔΔG ~ 4 kcal/mol
• Developing -ve charge (on oxygen) in TS is no longer
stabilized
• Energy diagram?
• Other mutants:
– Tyr34Phe
– His48Gly
– These other mutations showed smaller decreases in ΔG
– All contribute in some way to stabilize TS
2)
Do Thr-40 & His-45 really bind / phosphates?
Thr 40  Ala ( 7000 fold)
His 45  Gly ( 300 fold)
 Both decelerate the reaction
Double mutant  300,000 fold slower!
A Chemical Model for Adenylate Reaction
Mimic the proximity effect in an enzyme with small organic
molecules:
O
O
O
O
O
O
P
O
NO2
O
P
O
O
O
+
-O
Rate is comparable to tyrosyl-adenylate formation
 unimolecular reaction
NO2
Detect by UV
• Step 2 leads to adenylate; CO2H group is now activated
• Once activated, tRNAtyr-OH can bind
Step 3:
• 3’-OH attacks acyl
adenylate
• -ve charge increases on
O of carbonyl  Hbonding stabilizes this
charge (more in TS than
in SM)
•  H-bonding (of Gln) is
“more important” for TS
OH
tRNA
X-ray Structure of tRNAGln
• Example of tRNA bound to
tRNA synthase (stable without
Gln)
• tRNA (red) binds to enzyme
via multiple H-bonds
• 3’-OH oriented close to ATP
(consistent with proposed
mechanism in tyrosyl-tRNA)
ATP
3-’OH
Unique Role of Methionine
• Recall, Methionine is the 1st amino acid in a
peptide/protein (start codon)
• As seen previously, Met is also formylated
From N-formyltetrahydrofolate
O
fMet
S
tRNA
S
OH
H
O
HO2C
NH2
NH2
tRNA
O
Met
Met-tRNA fMet
S
X
O
tRNA
O
N
H
O
H
fMet-tRNA fMet
protected
Reaction is catalysed by
becoming pseudintramolecular (recall DNA
template synthesis):
Ribosome holds pieces
together  Ribosome is
cellular “workbench”
Protection with formyl
group allows
condensation one way
around only (only one
nucleophile)
tRNAfMet falls off
P site
Dipeptide moves over to
P site