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-bondingsalt bridgeIonicDipole-dipoleVan der Waals

CH2OH moieties are different because of non-equivalent binding sites in the enzyme

Enantioselection by an Enzyme

OH OH

OH

OH OPO3

OHH

* 2-glycerol kinase

achiral 3-glycerol phosphate

Three-Point Binding Model - Enantiospecificity

• Only one enantiomer binds to enzyme & is involved in reaction

B D

A

C B D

A

C

dbc

dbc

3 interactions

good fit, high H

C D

A

B

A D

B

C

dbc

poor fit, low H

1 interaction

dbc

poor fit, low H

2 interactions

With the other enantiomer…

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:

Km (Michaelis constant): small value indicates high affinity for substrate

Kbinding ( ~ 1/Km)

Strong binding → K > 1

ΔG= -RT ln KΔG must be –ve

E S+ E.S (Michaelis Complex)K

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 non-covalent interactions (i.e. 3 points)

Back to tyrosyl-tRNA synthase:

NH3

O

O

OH

P

O

O

Adenosine O P

O

O

O OP

O

O

NH3

O

O

OH

P

O

Adenosine

O

O

R

OHOH

B

O

R

OHO

B

NH3

O

OH

tRNA Tyr

+

+

+

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

OH

Tyrosine + ATP + Enz Enz.Tyr .ATP Enz.Tyr--AMP + H2P2O73-

(released)

bind tRNA

Enz.Tyr--AMP . tRNAEnz.Tyr--OtRNA + AMP

(released)

Enz + Tyr--OtRNA

Can isolate this complex in the absence of tRNA

Tyrosyl-tRNA Synthase.tyr-adenylate

*

* ** Main chain contacts

Tyr specificity

Binding AAs

3 point binding enantiospecificity

Bind ATP

ATP, not dATP

*

* ** Main chain contacts

-O

P

P

Orient PO4 towards CO2

-

Increase P+

• 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 PO4

2- () giving pentacoordinate P (trigonal bipyramidal) intermediate

O

O

P

O

-O O-O

AdO

O

P

O

-OO

-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 H-bonds 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

OP O

O

O

O

NO2 OP

O

O

O

O

-O NO2

+

Detect by UVRate is comparable to tyrosyl-adenylate formation unimolecular reaction

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

OHtRNA

• Step 2 leads to adenylate; CO2H group is now activated

• Once activated, tRNAtyr-OH can bind

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)

3-’OH

ATP

Unique Role of Methionine

• Recall, Methionine is the 1st amino acid in a peptide/protein (start codon)

• As seen previously, Met is also formylated

NH2

S

HO2CNH2

S

O

OtRNA N

H

S

O

OtRNA H

OtRNA OH

H X

O

Met Met-tRNA fMetfMet-tRNA fMet

fMet

From N-formyltetrahydrofolate

protected

Protection with formyl group allows condensation one way around only (only one nucleophile)

Reaction is catalysed by becoming pseud-intramolecular (recall DNA template synthesis): Ribosome holds pieces together Ribosome is cellular “workbench”

tRNAfMet falls off P site

Dipeptide moves over to P

site

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

dTdAdC

dAdTdA

dGdCdT

5'

3'

DNA

RNA polymerase

Transcription

AUG

UAU

CGA

mRNA

UAC

AUA

CODON Met

Tyr

P site

A site

Rxn & translocation

AUA

GCU

AGC

UAU

CGA

mRNA

MetTyr

Arg

P site

A site

Met Tyr ArgH NH

O

O

O

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 Ǻ of “active site”• rRNA (catalytic RNA) has been proposed :

N

R

O

O

H

H

tRNA

N

N

NN

NH2

O

pep

O

tRNA

NH

R

O

O

tRNA

O

tRNAO

pep

H

N

N

NN

NH2

NH

R

O

O

tRNA

O

pep

N

N

NN

NH2

OH

A2486

+

tRNA

Adenosine from rRNA

• 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 3’OH:

• OH → H

• OH → F

Both substitutions reduce rate by 106!

adenosine

Why the Reduction in the Rate?

Accounts for most of rate acceleration e.g. of catalytic RNA & substrate catalysis

O

O

O O

A76

tRNA

O

H

O

OA76

tRNA

O O

ONH2

R

P site A site