Title: The Chemistry of Protein Catalysis
1The Chemistry of Protein Catalysis
- John Mitchell
- University of St Andrews
2The MACiE Database
Mechanism, Annotation and Classification in
Enzymes. http//www.ebi.ac.uk/thornton-srv/databas
es/MACiE/
Gemma Holliday, Daniel Almonacid, Noel OBoyle,
Janet Thornton, Peter Murray-Rust, Gail
Bartlett, James Torrance, John Mitchell
G.L. Holliday et al., Nucl. Acids Res., 35,
D515-D520 (2007)
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4Enzyme Nomenclature and Classification
5The EC Classification
- Deals with overall reaction, not mechanism
- Reaction direction arbitrary
- Cofactors and active site residues ignored
- Doesnt deal with structural and sequence
information - However, it was never intended to do so
6A 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
7MACiE Database
Mechanism, Annotation and Classification in
Enzymes. http//www.ebi.ac.uk/thornton-srv/databas
es/MACiE/
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10Global Usage of MACiE
11MACiE Entries
12Difficulties 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.
13Classifying Related Enzymes Phosphoinositide-spe
cific Phospholipases C
Eukaryotic (rat) Cell Signalling Multidomain Catal
ytic 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/s
low final hydrolysis Disfavours
bisphosphate Acid-base mechanism Not calcium
dependent
14Difficulties 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.
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16MACiE Mechanisms are Sourced from the Literature
17Coverage of MACiE
Representative based on a non-homologous
dataset, and chosen to represent each available
EC sub-subclass.
18EC 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.
19EC Coverage of MACiE
20Repertoire 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)
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23Repertoire of Enzyme Catalysis
Enzyme chemistry is largely nucleophilic
24Repertoire of Enzyme Catalysis
Enzyme chemistry is largely nucleophilic
25Repertoire of Enzyme Catalysis
26Repertoire of Enzyme Catalysis
27Repertoire of Enzyme Catalysis
28Repertoire of Enzyme Catalysis
29Repertoire of Enzyme Catalysis
We do see a few steps corresponding to well-known
organic reactions but these are the exception.
30Repertoire of Enzyme Catalysis
31We divide residue roles into three
categories Reactant Covalently involved in the
reaction step, Spectator Stabilisation,
activation, steric roles, Interaction Hydrogen
bonding etc.
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33Residue Catalytic Propensities
34Residue Catalytic Functions
35Convergent Evolution of Enzyme Function
N.M. OBoyle et al., J. Molec. Biol., 368,
1484-1499 (2007)
D.E. Almonacid et al., PLoS Computational
Biology, accepted
36We use a combination of bioinformatics
chemoinformatics to identify similarities between
enzyme-catalysed reaction mechanisms
37Similarity of Overall Reactions Compare Bond
Changes
38Similarity of Mechanisms Compare Steps
Just like sequence alignment!
We can measure their similarity
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40Carrying out an analysis of pairwise similarity
of reactions in MACiE ...
41Find only a few similar pairs
42Identify convergent evolution
43Check MACiE for duplicates!
44Mechanistic similarity is only weakly related to
proximity in the EC classification
45EC in common
0 -.-.-.-
1 c.-.-.-
2 c.s.-.-
3 c.s.ss.-
46Similarity 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).
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5043 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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52One third of analogous pairs with significantly
similar overall reactions have significantly
similar mechanisms.
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54For analogous pairs, we find that mechanistic
similarity is less than overall similarity
(almost always) these lie in the lower triangle.
55Conclusions 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).
56ACKNOWLEDGEMENTS
Dr Gemma Holliday Dr Daniel Almonacid Dr Noel
OBoyle Prof. Janet Thornton (EBI) Prof. Patsy
Babbitt (UCSF) Dr Peter Murray-Rust Dr Florian
Nigsch
57ACKNOWLEDGEMENTS