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

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

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

5. The EC Classification  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  Deals with overall reaction, not mechanism

6. 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

7. MACiE Mechanism, Annotation and Classification in Enzymes. http://www.ebi.ac.uk/thornton-srv/databases/MACiE/ MACiE Database

10. Global Usage of MACiE

11. MACiE Entries

12. 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.

13. 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

14. 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.

16. MACiE Mechanisms are Sourced from the Literature

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

18. EC Coverage of MACiE Representative – based on a non-homologous dataset, and chosen to represent each available EC sub-subclass. Structures exist for: 6 EC 1.-.-.- 57 EC 1.2.-.- 194 EC 1.2.3.- 1547 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

19. EC Coverage of MACiE

20. 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)

23. Repertoire of Enzyme Catalysis Enzyme chemistry is largely nucleophilic

24. Repertoire of Enzyme Catalysis Enzyme chemistry is largely nucleophilic

25. Protontransfer Ad N 2 E1 SN2SN2SN2SN2E2 RadicalreactionTautom.Others Repertoire of Enzyme Catalysis

29. We do see a few steps corresponding to well- known organic reactions; but these are the exception.

30. Repertoire of Enzyme Catalysis

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

33. Residue Catalytic Propensities

34. Residue Catalytic Functions

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

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

37. Similarity of Overall Reactions: Compare Bond Changes

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

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

41. Find only a few similar pairs

42. Identify convergent evolution

43. Check MACiE for duplicates!

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

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

46. 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).

50. 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.

52. One third of analogous pairs with significantly similar overall reactions have significantly similar mechanisms.

54. For analogous pairs, we find that mechanistic similarity is less than overall similarity (almost always); these lie in the lower triangle.

55. Conclusions for Analogous Enzymes Conservation of EC sub-subclass does not imply quantitative reaction similarity. One third of analogous pairs with significantly similar overall reactions have significantly similar mechanisms. Mechanistic similarity is less than overall similarity (unlike homologues).

56. ACKNOWLEDGEMENTS Dr Gemma Holliday Dr Daniel Almonacid Dr Noel O’Boyle Prof. Janet Thornton (EBI) Prof. Patsy Babbitt (UCSF) Dr Peter Murray-Rust Dr Florian Nigsch

57. ACKNOWLEDGEMENTS Cambridge Overseas Trust