The Chemistry of Protein
Catalysis
John Mitchell
University of St Andrews
The MACiE Database
Mechanism, Annotation and Classification in Enzymes.
http://www.ebi.ac.uk/thornton-srv/databases/MACiE/
Gemma Holliday, Daniel Almonacid, Noel O’Boyle,
Janet Thornton, Peter Murray-Rust, Gail Bartlett,
James Torrance, John Mitchell
G.L. Holliday et al., Nucl. Acids Res., 35, D515-D520 (2007)
Enzyme Nomenclature and
Classification
EC Classification
Class
Subclass
Sub-subclass
Serial number
The EC Classification
Deals with overall reaction, not mechanism
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
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
MACiE Database
Mechanism, Annotation and Classification in Enzymes.
http://www.ebi.ac.uk/thornton-srv/databases/MACiE/
Global Usage of MACiE
MACiE Entries
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.
Classifying Related Enzymes:
Phosphoinositide-specific Phospholipases C
Eukaryotic (rat)
Cell Signalling
Prokaryotic (B. cereus)
Virulence factor
Multidomain
Catalytic TIM Barrel
EC 3.1.4.11
Single domain
Catalytic TIM Barrel
EC 4.6.1.13
Hydrolase
Final hydrolysis step
Prefers bisphosphate
Acid-base mechanism
Lyase
No/slow final hydrolysis
Disfavours bisphosphate
Acid-base mechanism
Calcium dependent
Not calcium dependent
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.
MACiE Mechanisms are Sourced from the Literature
Coverage of MACiE
Representative – based on a non-homologous dataset,
and chosen to represent each available EC sub-subclass.
EC Coverage of MACiE
Structures exist for:
MACiE covers:
6 EC 1.-.-.-
6 EC 1.-.-.-
57 EC 1.2.-.-
54 EC 1.2.-.-
194 EC 1.2.3.-
165 EC 1.2.3.-
1450 EC 1.2.3.4
249 EC 1.2.3.4
Representative – based on a non-homologous dataset,
and chosen to represent each available EC sub-subclass.
EC Coverage of MACiE
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)
Number of steps in MACiE
Repertoire of Enzyme Catalysis
140
Intramolecular
120
Bimolecular
Unimolecular
Enzyme chemistry
is largely nucleophilic
100
80
60
40
20
0
Heterolytic
Elimination
Homolytic
Elimination
Electrophilic
Addition
Nucleophilic
Addition
Homolytic
Addition
Reaction Types
Electrophilic
Substitution
Nucleophilic
Substitution
Homolytic
Substitution
Repertoire of Enzyme Catalysis
Enzyme chemistry
is largely nucleophilic
Repertoire of Enzyme Catalysis
450
400
Number of steps in MACiE
350
300
250
200
150
100
50
0
Proton
transfer
AdN2
E1
SN2
E2
Reaction Types
Radical
reaction
Tautom.
Others
Repertoire of Enzyme Catalysis
Repertoire of Enzyme Catalysis
Repertoire of Enzyme Catalysis
Repertoire of Enzyme Catalysis
We do see a few steps
corresponding to wellknown organic reactions;
but these are the
exception.
Repertoire of Enzyme Catalysis
We divide residue roles into three categories:
Reactant: Covalently involved in the reaction step,
Spectator: Stabilisation, activation, steric roles,
Interaction: Hydrogen bonding etc.
Residue Catalytic Propensities
Residue Catalytic Functions
Convergent Evolution of
Enzyme Function
N.M. O’Boyle et al., J. Molec. Biol., 368, 1484-1499 (2007)
D.E. Almonacid et al., PLoS Computational Biology, accepted
We use a combination of bioinformatics &
chemoinformatics to identify similarities between
enzyme-catalysed reaction mechanisms
Similarity of Overall Reactions: Compare Bond Changes
Similarity of Mechanisms: Compare Steps
Just like sequence
alignment!
We can measure
their similarity …
Carrying out an analysis of pairwise
similarity of reactions in MACiE ...
Find only a few similar pairs
Identify convergent evolution
Check MACiE for duplicates!
Mechanistic similarity is only weakly related
to proximity in the EC classification
EC in common
0 -.-.-.1 c.-.-.2 c.s.-.3 c.s.ss.-
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).
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.
One third of analogous pairs with significantly similar
overall reactions have significantly similar
mechanisms.
For analogous pairs, we find that
mechanistic similarity is less than overall
similarity (almost always); these lie in the
lower triangle.
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).
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
ACKNOWLEDGEMENTS
Cambridge Overseas
Trust