The Chemistry of Protein Catalysis

1. The Chemistry of Protein Catalysis John Mitchell

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 (EBI), Peter Murray-Rust, Gail Bartlett (EBI), 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 • Only deals with overall reaction • 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. Coverage of MACiE Representative – based on a non-homologous dataset, and chosen to represent each available EC sub-subclass.

8. Coverage of MACiE Structures exist for: 6 EC 1.-.-.- 56 EC 1.2.-.- 184 EC 1.2.3.- 1312 EC 1.2.3.4 MACiE covers: 6 EC 1.-.-.- 53 EC 1.2.-.- 156 EC 1.2.3.- 199 EC 1.2.3.4 Representative – based on a non-homologous dataset, and chosen to represent each available EC sub-subclass.

9. Repertoire of Enzyme Catalysis G.L. Holliday et al.,J. Molec. Biol., 372, 1261-1277 (2007) G.L. Holliday et al.,J. Molec. Biol., accepted (2009)

12. Repertoire of Enzyme Catalysis Enzyme chemistry is largely nucleophilic

13. Repertoire of Enzyme Catalysis Enzyme chemistry is largely nucleophilic

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

15. Repertoire of Enzyme Catalysis

16. Repertoire of Enzyme Catalysis

17. Repertoire of Enzyme Catalysis

18. Repertoire of Enzyme Catalysis

19. Residue Catalytic Propensities

20. Residue Catalytic Functions

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

22. … we align the steps of chemical reactions. Just like sequence alignment! We can measure their similarity …

23. Find only a few similar pairs

24. Identify convergent evolution

25. Check MACiE for duplicates

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

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

28. Evolution of Enzyme Function D.E. Almonacid et al., to be published

29. EC is our Functional Classification Chemical reaction Enzyme Commission (EC) Nomenclature, 1992, Academic Press, San Diego, 6th Edition

30. Enzyme catalysis databases G.L. Holliday et al., Nucleic Acids Res.,35, D515 (2007) S.C. Pegg et al., Biochemistry, 45, 2545 (2006) N. Nagano, Nucleic Acids Res., 33, D407 (2005)

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

32. Coverage of SFLD Based on a few evolutionarily related families

33. Coverage of EzCatDB But without mechanisms.

34. Domains Work with domains - evolutionary & structural units of proteins. Map enzyme catalytic mechanisms to domains to quantify convergent and divergent functional evolution of enzymes.

35. CATH is our Structural Classification Orengo, C. A., et al. Structure, 1997, 5, 1093

36. Results: Convergent Evolution Numbers of CATH code occurrences per EC number c.s.-.- c.s.ss.- c.s.ss.sn c.-.-.- C 3.17 1.73 1.38 1.11 A 11.00 3.27 1.93 1.60 T 28.00 4.89 2.24 1.19 H 2.46 38.33 5.80 1.22 2.46CATH/EC reaction Convergent Evolution

37. Results:Convergent Evolution Numbers of CATH code occurrences per EC number c.s.-.- c.s.ss.- c.s.ss.sn c.-.-.- C 3.17 1.73 1.38 1.11 A 11.00 3.27 1.93 1.60 T 28.00 4.89 2.24 1.19 H 2.46 38.33 5.80 1.22 2.46CATH/EC reaction: Convergent Evolution An average reaction has evolved independently in 2.46superfamilies

38. Results: Divergent Evolution EC reactions/CATH H 1.20 1.36 T 1.36 1.79 2.08 3.05 A 3.14 7.00 10.48 17.90 C 4.75 19.50 39.25 90.00 c.-.-.- c.s.-.- c.s.ss.- c.s.ss.sn 1.46 2.05 1.46EC reactions/CATHDivergent Evolution databaseentries/CATH 2.18

39. Results: Divergent Evolution EC reactions/CATH H 1.20 1.36 T 1.36 1.79 2.08 3.05 A 3.14 7.00 10.48 17.90 C 4.75 19.50 39.25 90.00 c.-.-.- c.s.-.- c.s.ss.- c.s.ss.sn 1.46 2.05 1.46EC reactions/CATH: Divergent Evolution An average superfamily has evolved 1.46 different reactions databaseentries/CATH 2.18

40. The Future …

41. (1) Molecular Evolution

42. Now we want to evolve chemical reactions in silico across chemical, or EC, space. 1. To understand and rationalise convergent and divergent biochemical evolution; 2. To better relate protein structure and function; 3. To understand the influence on networks of coupled reactions.

43. (2) Understanding Protein Structure • We seek to understand the influence of folding pathway on protein structure over all time scales (including the evolutionary one).

44. 44

45. Protein Folding Funnel Energy Landscape 45

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

48. ACKNOWLEDGEMENTS Cambridge Overseas Trust