The Chemistry of Protein
Catalysis
John Mitchell
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 (EBI), Peter Murray-Rust, Gail Bartlett (EBI),
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
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
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/
Coverage of MACiE
Representative – based on a non-homologous dataset,
and chosen to represent each available EC sub-subclass.
Coverage of MACiE
Structures exist for:
MACiE covers:
6 EC 1.-.-.-
6 EC 1.-.-.-
56 EC 1.2.-.-
53 EC 1.2.-.-
184 EC 1.2.3.-
156 EC 1.2.3.-
1312 EC 1.2.3.4
199 EC 1.2.3.4
Representative – based on a non-homologous dataset,
and chosen to represent each available EC sub-subclass.
Repertoire of Enzyme Catalysis
G.L. Holliday et al., J. Molec. Biol., 372, 1261-1277 (2007)
G.L. Holliday et al., J. Molec. Biol., accepted (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
Residue Catalytic Propensities
Residue Catalytic Functions
We use a combination of bioinformatics &
chemoinformatics to identify similarities between
enzyme-catalysed reaction mechanisms
… we align the steps of chemical reactions.
Just like sequence
alignment!
We can measure
their similarity …
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.-
Evolution of Enzyme Function
D.E. Almonacid et al., to be published
EC is our Functional Classification
Chemical reaction
Enzyme Commission (EC) Nomenclature, 1992, Academic Press, San Diego, 6th Edition
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)
Coverage of MACiE
Representative – based on a non-homologous dataset,
and chosen to represent each available EC sub-subclass.
Coverage of SFLD
Based on a few evolutionarily related families
Coverage of EzCatDB
But without mechanisms.
Domains
Work with domains - evolutionary & structural units of proteins.
Map enzyme catalytic mechanisms to domains to quantify
convergent and divergent functional evolution of enzymes.
CATH is our Structural Classification
Orengo, C. A., et al. Structure, 1997, 5, 1093
Results: Convergent Evolution
Numbers of CATH code occurrences per EC number
c.s.-.c.s.ss.c.s.ss.sn
c.-.-.-
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
Results: Convergent Evolution
Numbers of CATH code occurrences per EC number
c.s.-.c.s.ss.c.s.ss.sn
c.-.-.-
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
Results: Divergent Evolution
EC reactions/CATH
c.-.-.-
C
4.75
A
3.14
T
1.36
H
1.20
c.s.-.-
19.50
7.00
1.79
1.36
c.s.ss.-
39.25
10.48
2.08
1.46
c.s.ss.sn
90.00
17.90
3.05
2.05
1.46 EC reactions/CATH
database entries/CATH
Divergent
Evolution
2.18
Results: Divergent Evolution
EC reactions/CATH
c.-.-.-
C
4.75
A
3.14
T
1.36
H
1.20
c.s.-.-
19.50
7.00
1.79
1.36
c.s.ss.-
39.25
10.48
2.08
1.46
c.s.ss.sn
90.00
17.90
3.05
2.05
1.46 EC reactions/CATH: Divergent Evolution
database
An average superfamily
hasentries/CATH
evolved 1.46 different reactions
2.18
The Future …
(1) Molecular Evolution
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.
(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
Protein Folding Funnel
Energy Landscape
45
ACKNOWLEDGEMENTS
Dr Gemma Holliday
Dr Daniel Almonacid
Dr Noel O’Boyle
Prof. Janet Thornton (EBI)
Dr Peter Murray-Rust
Dr Florian Nigsch
ACKNOWLEDGEMENTS
Cambridge Overseas
Trust