1. The Chemistry of Protein Catalysis
John Mitchell

2. The MACiE Database
Mechanism, Annotation and Classification in Enzymes.
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. Enzyme Nomenclature and Classification
EC Classification
Class
Subclass
Sub-subclass
Serial number

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. Mechanism, Annotation and Classification in Enzymes.
MACiE Database
Mechanism, Annotation and Classification in Enzymes.

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.-.-
MACiE covers:
6 EC
53 EC
156 EC
199 EC
1312/184~7
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, (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. Repertoire of Enzyme Catalysis
Proton
transfer
AdN2 E1
SN2 E2
Radical
reaction
Tautom.
Others

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. Just like sequence alignment!
… 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 
 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. Based on a few evolutionarily related families
Coverage of SFLD
Based on a few evolutionarily related families

33. But without mechanisms.
Coverage of EzCatDB
But without mechanisms.

34. 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.-.-.-
c.s.-.-
c.s.ss.-
c.s.ss.sn 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
38.33
5.80
2.46
1.22
2.46 CATH/EC reaction
Convergent Evolution

37. Numbers of CATH code occurrences per EC number
Results: Convergent Evolution
Numbers of CATH code occurrences per EC number
c.-.-.-
c.s.-.-
c.s.ss.-
c.s.ss.sn 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
38.33
5.80
2.46
1.22
2.46 CATH/EC reaction: Convergent Evolution
An average reaction has evolved independently in 2.46 superfamilies

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

39. Results: Divergent Evolution database entries/CATH
EC reactions/CATH C
4.75
19.50
39.25
90.00 A
3.14
7.00
10.48
17.90 T
1.36
1.79
2.08
3.05 H
1.20
1.36
c.-.-.-
c.s.-.-
c.s.ss.-
c.s.ss.sn
1.46
2.05
1.46 EC reactions/CATH: Divergent Evolution
An average superfamily has evolved 1.46 different reactions
database entries/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. 5788 (~12%) of PDB come from Structural Genomics44

45. Protein Folding Funnel
Energy Landscape
5788 (~12%) of PDB come from Structural Genomics 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