The Chemistry of Protein Catalysis

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The Chemistry of Protein Catalysis

• John Mitchell
• University of St Andrews

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The 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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Enzyme Nomenclature and Classification
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The 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

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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

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MACiE Database
Mechanism, Annotation and Classification in
Enzymes. http//www.ebi.ac.uk/thornton-srv/databas
es/MACiE/
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Global Usage of MACiE
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MACiE Entries
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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.

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Classifying 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
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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.

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MACiE Mechanisms are Sourced from the Literature
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Coverage of MACiE
Representative based on a non-homologous
dataset, and chosen to represent each available
EC sub-subclass.
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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.
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EC Coverage of MACiE
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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)
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Repertoire of Enzyme Catalysis
Enzyme chemistry is largely nucleophilic
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Repertoire of Enzyme Catalysis
Enzyme chemistry is largely nucleophilic
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Repertoire of Enzyme Catalysis
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Repertoire of Enzyme Catalysis
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Repertoire of Enzyme Catalysis
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Repertoire of Enzyme Catalysis
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Repertoire of Enzyme Catalysis
We do see a few steps corresponding to well-known
organic reactions but these are the exception.
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Repertoire of Enzyme Catalysis
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We 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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Residue Catalytic Propensities
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Residue Catalytic Functions
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Convergent 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
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We use a combination of bioinformatics
chemoinformatics to identify similarities between
enzyme-catalysed reaction mechanisms
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Similarity of Overall Reactions Compare Bond
Changes
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Similarity of Mechanisms Compare Steps
Just like sequence alignment!
We can measure their similarity
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Carrying out an analysis of pairwise similarity
of reactions in MACiE ...
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Find only a few similar pairs
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Identify convergent evolution
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Check MACiE for duplicates!
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Mechanistic similarity is only weakly related to
proximity in the EC classification
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EC in common
0 -.-.-.-
1 c.-.-.-
2 c.s.-.-
3 c.s.ss.-
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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).

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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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One third of analogous pairs with significantly
similar overall reactions have significantly
similar mechanisms.
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For analogous pairs, we find that mechanistic
similarity is less than overall similarity
(almost always) these lie in the lower triangle.
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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).

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