Insights into Protein BiosynthesisEntropic Origin of Catalytic Power?

Department of Chemistry

Senior Seminar

April 2, 2009

Luigi J. AlvaradoBiochemistry, B.S.Class of 2009

Outline

I. Protein TranslationII. The RibosomeIII. Chemistry of Peptide-

bond FormationIV. Entropic

Phenomenon?V. Other factorsVI. ConclusionsVII. Aknowledgements

I. PROTEIN TRANSLATION

http://www.youtube.com/watch?v=Jml8CFBWcDs&feature=relatedhttp://www.youtube.com/watch?v=5bLEDd-PSTQ

II. Ribosome

• 2.5 MDa/ 4 MDa• rRNA + Protein• Subunits, Domains• A-, P-, E-sites• Tunnels• Peptidyl TransferCenter (PTC)

II. Ribosome (cont’d) - PTC

• Domain V of 23S rRNA• No proteins within 15Å• Provides suitable

environment• tRNA-binding NTs:• G2251, G2252, A2448,

A2450, G2455, U2506, G2583, U2585, and A2602.

• 2-fold symmetry– Synchronized rotation

duet (45° 180°)– Geometry between the

moieties

• Solvent reorganization

Bashan et al. Mol. Cell 2003, 11, 91-102.

II. Ribosome (cont’d) - PTC

Bashan et al. Mol. Cell 2003, 11, 91-102.

III. Chemistry of Peptide-Bond Formation

Ribosomal Reference

Six-member Transition Intermediate

III. Chemistry of Peptide-Bond Formation: Ribosomal

Rodnina et al. Biochem. Soc. Trans. 2005, 33, 493-498.

• Nucleophilic attack• Free-tRNA + pept-tRNA• Regio- and stereo-specificity• Methods: Quench-flow assays • 1967: Fragment Rxns N-blocked aminoacylated

oligoNT (CCA-fMet) and Pmn• 2002: pept-tRNA + CPmn

III. Chemistry of Peptide-Bond Formation: Ribosomal

Sievers et al. Proc. Natl. Acad. Sci. 2004, 101, 7897-7901.

III. Chemistry of Peptide-Bond Formation: Reference

Schr

oede

r, G

; Wol

fend

en, R

. Bio

chem

istr

y 20

07, 4

6, 4

037-

4044

• Ester aminolysis• Methods: 1H-NMR• Pseudo-first and second

order kinetics• Exclusive attack of the

conjugate base of glycinamide.

• Polar solvent (+)• Ionic strength (0)

III. Chemistry of Peptide-Bond Formation: Reference

N-fPhe-glycinamide

N-fPhe-TFE

N-fPhe

Schr

oede

r, G

; Wol

fend

en, R

. Bio

chem

istr

y 20

07, 4

6, 4

037-

4044

Six-Member Transition Intermediate III. Chemistry of Peptide-Bond Formation

Weinger, J.; Strobel, S. Biochemistry 2006, 45, 5939-5948.

• ΔH‡ = Ea – RT

• ΔG‡ = -RTln[(kcat/(KMh))/(kB*T)]

• ΔG‡ = ΔH‡ - TΔS‡

III. Chemistry of Peptide-Bond FormationKinetics and Thermodynamics

Sievers et al. Proc. Natl. Acad. Sci. 2004, 101, 7897-7901

III. Chemistry of Peptide-Bond Formation

Kinetics and Thermodynamics

T = 25°CpH = 7.5

Schroeder, G; Wolfenden, R. Biochemistry 2007, 46, 4037-4044

M-1s-1 Kcal/mol

1X103 / 3X10-5 = 3X107

Ribosomal ReferenceRate Enhancement

IV. Entropic Phenomenon?

• ΔΔG‡ ~ -9 kcal/mol• ΔΔH‡ ~8 kcal/mol• ΔTΔS‡ ~18 kcal/mol

• What increases the TΔS‡?– Juxtaposition of substrates– Desolvation of PTC

Methods:

MD/EVB simulationsLangevin Dipole solvent, COSMO, and Restraint Release

Explanations:

Proton shuttle modelH-bond network

IV. Entropic Phenomenon? Juxtaposition of the Substrates

Beringer, M.; Rodnina, M. Mol. Cell 2007, 26, 311-321

IV. Entropic Phenomenon?

Desolvation of the PTC

Sharma et al. Biochemistry 2005, 44, 11307-11314

Solvation Orientational

Bring reactants to same solvent cage

V. Other Factors

H-bond Networks Pre-set Electr. Environ.

Beringer, M.; Rodnina, M. Mol. Cell 2007, 26, 311-321

VI. Conclusions

Schroeder, G; Wolfenden, R. Biochemistry 2007, 46, 4037-4044

VI. Conclusions

• The Ribosome is an entropy trap• Mechanism of catalysis is not driven via

ΔH‡

• Ribosome provides perfect environment• 6-member TI ↔ Proton Shuttle mech.• Other factors’ influence

Aknowledgements

• Dr. I. Kovach• Department of Chemistry Faculty• Class of 2009 – 2010 – 2011• Various researchers

I Chemistry