Three-Point Binding Model

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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

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Enantioselection by an Enzyme
CH2OH moieties are different because of
non-equivalent binding sites in the enzyme
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Three-Point Binding Model - Enantiospecificity

• Only one enantiomer binds to enzyme is involved
  in reaction

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With the other enantiomer
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• ? 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

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Binding

• The cost of binding
• Km (Michaelis constant) small value indicates
  high affinity for substrate
• ? Kbinding ( 1/Km)
• Strong binding ? K gt 1
• ?G -RT ln K
• ?G must be ve

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• ?Gbinding ?Hbinding- T?Sbinding
• For 2 molecules in, 1 out ?S is ve
• ? (-T?S) term is ve
• Entropy disfavors binding of substrate to enzyme
• To get good binding, need ve ?H (i.e. bond
  formation)
• Each non-covalent interaction is small (H-bond
  5 kcal/mol), but still gives a ve ?H
• Enzymes use many FGs to sum up many weak
  non-covalent interactions (i.e. 3 points)

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• Back to tyrosyl-tRNA synthase

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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

Can isolate this complex in the absence of tRNA
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Tyrosyl-tRNA Synthase.tyr-adenylate
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Bind ATP
Binding AAs

3 point binding enantiospecificity
ATP, not dATP
Tyr specificity


Main chain contacts
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Orient ? PO4 towards CO2-

Increase P?


Main chain contacts
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• 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

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• 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 H-bonds are formed in TS than in trig.
Bipyramidal intermediate
?
Lower TS energy ? accelerate collapse of
intermediate
Gln195
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Tests of Mechanism

• Site-directed mutagenesis
• Replace Gln195 with Gly ? (Gln195Gly)
• Rate slows by gt 1000 fold
• ??G? 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
  ?G
• All contribute in some way to stabilize TS

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• 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!

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A Chemical Model for Adenylate Reaction

• Mimic the proximity effect in an enzyme with
  small organic molecules

Rate is comparable to tyrosyl-adenylate formation
? unimolecular reaction
Detect by UV
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• 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 ? H-bonding
  stabilizes this charge (more in TS than in SM)
• ? H-bonding (of Gln) is more important for TS

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X-ray Structure of tRNAGln
3-OH

• 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
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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
protected
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Reaction is catalysed by becoming
pseud-intramolecular (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
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Control of Sequence

• mRNA (messenger RNA) made by copying sequence of
  DNA in gene
• Goes to ribosome, along with rRNA (ribosomal
  RNA-part of ribosome structure) tRNA (with AAs
  attached)
• In mRNA, 3 nucleotides of specific sequence
  encode 1 amino acid (CODON)
• R-tRNAR has 3 nucleotides complementary through
  base pairing to the codon for R
• Specific binding at A site
• Codons for start stop control the final protein
  length

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P site
CODON
A site
Rxn translocation
P site
Tyr
Met
A site
Arg
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Catalysis of Reaction?

• Synthesis on ribosome is faster by 107 than rxn
  without ribosome
• Peptide formation is not catalyzed by protein ?
  no protein within 20 ? of active site
• rRNA (catalytic RNA) has been proposed

Adenosine from rRNA
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• However, modification of bases has shown little
  effect on catalytic activity (2-fold decrease)
• May be the 2-OH (of tRNA) at last nucleotide on
  P site i.e., the substrate! (see Nature
  Struct. Mol. Biol. (2004), 11, p 1101

• Modified sugar at 3OH
• OH ? H
• OH ? F

Both substitutions reduce rate by 106!
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adenosine
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Why the Reduction in the Rate?
P site
A site
Accounts for most of rate acceleration ? e.g. of
catalytic RNA substrate catalysis