the chemistry of protein catalysis
DESCRIPTION
The Chemistry of Protein Catalysis. John Mitchell University of St Andrews. The MACiE Database. M echanism, A nnotation and C lassification i n E nzymes . http://www.ebi.ac.uk/thornton-srv/databases/MACiE/. Gemma Holliday, Daniel Almonacid, Noel O’Boyle, - PowerPoint PPT PresentationTRANSCRIPT
The Chemistry of Protein The Chemistry of Protein CatalysisCatalysis
John Mitchell
University of St Andrews
MMechanism, AAnnotation and CClassification iin EEnzymes.http://www.ebi.ac.uk/thornton-srv/databases/MACiE/
The MACiE DatabaseThe MACiE Database
G.L. Holliday et al., Nucl. Acids Res., 35, D515-D520 (2007)
Gemma Holliday, Daniel Almonacid, Noel O’Boyle,
Janet Thornton, Peter Murray-Rust, Gail Bartlett,
James Torrance, John Mitchell
Enzyme Nomenclature and Enzyme Nomenclature and ClassificationClassificationEC ClassificationEC Classification
Class
Subclass
Sub-subclass
Serial number
The EC ClassificationThe EC Classification
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
Deals with overall reaction, not mechanism
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
MMechanism, AAnnotation and CClassification iin EEnzymes.http://www.ebi.ac.uk/thornton-srv/databases/MACiE/
MACiE DatabaseMACiE Database
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
Multidomain
Catalytic 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/slow final hydrolysis
Disfavours bisphosphate
Acid-base mechanism
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
Representative – based on a non-homologous dataset,and chosen to represent each available EC sub-subclass.
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
EC Coverage of MACiE
Repertoire of Enzyme CatalysisRepertoire 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)
Repertoire of Enzyme Catalysis
0
20
40
60
80
100
120
140
HeterolyticElimination
HomolyticElimination
ElectrophilicAddition
NucleophilicAddition
HomolyticAddition
ElectrophilicSubstitution
NucleophilicSubstitution
HomolyticSubstitution
Reaction Types
Num
ber
of
step
s in
MA
CiE
Intramolecular
Bimolecular
Unimolecular
Enzyme chemistry is largely nucleophilic
Repertoire of Enzyme Catalysis
Enzyme chemistry is largely nucleophilic
0
50
100
150
200
250
300
350
400
450
Reaction Types
Num
ber
of
ste
ps in M
ACiE
ProtonProtontransfertransfer
AdAdNN22 E1E1 SSNN22 E2E2 RadicalRadicalreactionreaction
Tautom.Tautom. OthersOthers
Repertoire of Enzyme Catalysis
Repertoire of Enzyme Catalysis
Repertoire of Enzyme Catalysis
Repertoire of Enzyme Catalysis
Repertoire of Enzyme Catalysis
We do see a few steps corresponding to well-known 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 Convergent Evolution of Enzyme FunctionEnzyme Function
D.E. Almonacid et al., PLoS Computational Biology, accepted
N.M. O’Boyle et al., J. Molec. Biol., 368, 1484-1499 (2007)
We use a combination of bioinformatics & chemoinformatics to identify similarities between enzyme-catalysed reaction mechanisms
Similarity of Overall Reactions: Compare Bond Changes
Just like sequence alignment!
We can measure their similarity …
Similarity of Mechanisms: Compare Steps
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).
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
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
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
Cambridge Overseas
Trust