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The Chemistry of Protein Catalysis

The Chemistry of Protein Catalysis. John Mitchell University of St Andrews. The MACiE Database. M echanism, A nnotation and C lassification i n E nzymes . http://www.ebi.ac.uk/thornton-srv/databases/MACiE/. Gemma Holliday, Daniel Almonacid, Noel O’Boyle,

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The Chemistry of Protein Catalysis

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  1. The Chemistry of Protein Catalysis John Mitchell University of St Andrews

  2. The MACiE Database Mechanism,AnnotationandClassificationin Enzymes. http://www.ebi.ac.uk/thornton-srv/databases/MACiE/ Gemma Holliday, Daniel Almonacid, Noel O’Boyle, Janet Thornton, Peter Murray-Rust, Gail Bartlett, James Torrance, John Mitchell G.L. Holliday et al., Nucl. Acids Res., 35, D515-D520 (2007)

  3. EC Classification Class Subclass Sub-subclass Serial number Enzyme Nomenclature and Classification

  4. The EC Classification • Deals with overall reaction, not mechanism • Reaction direction arbitrary • Cofactors and active site residues ignored • Doesn’t deal with structural and sequence information • However, it was never intended to do so

  5. A New Representation of Enzyme Reactions? • Should be complementary to, but distinct from, the EC system • Should take into account: • Reaction Mechanism • Structure • Sequence • Active Site residues • Cofactors • Need a database of enzyme mechanisms

  6. MACiE Database Mechanism,AnnotationandClassificationin Enzymes. http://www.ebi.ac.uk/thornton-srv/databases/MACiE/

  7. Global Usage of MACiE

  8. MACiE Entries

  9. Difficulties of Hierarchical Classification • Very similar mechanisms can end up in different first level classes. • In the case of phosphoinositide-specific phospholipases C, this is due to a slow final hydrolysis step occurring in one of the two enzymes.

  10. Classifying Related Enzymes:Phosphoinositide-specific Phospholipases C Eukaryotic (rat) Cell Signalling Multidomain Catalytic TIM Barrel EC 3.1.4.11 Hydrolase Final hydrolysis step Prefers bisphosphate Acid-base mechanism Calcium dependent Prokaryotic (B. cereus) Virulence factor Single domain Catalytic TIM Barrel EC 4.6.1.13 Lyase No/slow final hydrolysis Disfavours bisphosphate Acid-base mechanism Not calcium dependent

  11. Difficulties of Hierarchical Classification • Different mechanisms can occur with exactly the same EC number. • MACiE has six beta-lactamases, all with different mechanisms but the same overall reaction.

  12. MACiE Mechanisms are Sourced from the Literature

  13. Coverage of MACiE Representative – based on a non-homologous dataset, and chosen to represent each available EC sub-subclass.

  14. EC Coverage of MACiE Structures exist for: 6 EC 1.-.-.- 57 EC 1.2.-.- 194 EC 1.2.3.- 1450 EC 1.2.3.4 MACiE covers: 6 EC 1.-.-.- 54 EC 1.2.-.- 165 EC 1.2.3.- 249 EC 1.2.3.4 Representative – based on a non-homologous dataset, and chosen to represent each available EC sub-subclass.

  15. EC Coverage of MACiE

  16. Repertoire of Enzyme Catalysis G.L. Holliday et al.,J. Molec. Biol., 372, 1261-1277 (2007) G.L. Holliday et al.,J. Molec. Biol., 390, 560-577 (2009)

  17. Repertoire of Enzyme Catalysis Enzyme chemistry is largely nucleophilic

  18. Repertoire of Enzyme Catalysis Enzyme chemistry is largely nucleophilic

  19. Proton transfer AdN2 E1 SN2 E2 Radical reaction Tautom. Others Repertoire of Enzyme Catalysis

  20. Repertoire of Enzyme Catalysis

  21. Repertoire of Enzyme Catalysis

  22. Repertoire of Enzyme Catalysis

  23. Repertoire of Enzyme Catalysis We do see a few steps corresponding to well-known organic reactions; but these are the exception.

  24. Repertoire of Enzyme Catalysis

  25. We divide residue roles into three categories: Reactant: Covalently involved in the reaction step, Spectator: Stabilisation, activation, steric roles, Interaction: Hydrogen bonding etc.

  26. Residue Catalytic Propensities

  27. Residue Catalytic Functions

  28. Convergent Evolution of Enzyme Function N.M. O’Boyle et al., J. Molec. Biol., 368, 1484-1499 (2007) D.E. Almonacid et al., PLoS Computational Biology, accepted

  29. We use a combination of bioinformatics & chemoinformatics to identify similarities between enzyme-catalysed reaction mechanisms

  30. Similarity of Overall Reactions: Compare Bond Changes

  31. Similarity of Mechanisms: Compare Steps Just like sequence alignment! We can measure their similarity …

  32. Carrying out an analysis of pairwise similarity of reactions in MACiE ...

  33. Find only a few similar pairs

  34. Identify convergent evolution

  35. Check MACiE for duplicates!

  36. Mechanistic similarity is only weakly related to proximity in the EC classification

  37. EC in common 0 -.-.-.- 1 c.-.-.- 2 c.s.-.- 3 c.s.ss.-

  38. Similarity of Analogous Reactions • We take all possible pairs of analogous enzyme reactions from MACiE 2.3.9 • Analogous means that they carry out similar functions (EC 1.2.3.- conserved) ... • ... and that the enzymes are not homologous • We find 95 analogous pairs (convergent evolution).

  39. 43 out of 95 pairs that are analogous according to EC have no significant reaction or mechanistic similarity Shared EC sub-subclass and Bond Change based reaction similarity are quite different criteria.

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