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MACiE – a Database
of Enzyme Reaction
Mechanisms
Janet Thornton
EMBL-EBI
July 2006
Enzymes in Data Resources (2005)
No. enzymes Total No.
in DB
%-tage
UniProt
65,076
172,690
37.7
PDB (all entries)
PDB (non-redundant)
14,143
3,655
31,522
10,450
44.9
35.0
230
680
33.8
Reactome (human)
(via UniProt)
Roman Laskowski
Number of enzymes
Relating the number of enzymes to proteome size
Permissive set
KEGG assignments
Conservative set
human
mouse
fly
worm
Proteome size
Shiri Freilich
Increase in Number of Different Reactions (E.C.) in larger proteomes
E.C. 1
E.C. 4
E.C. 2
E.C. 5
E.C. 3
E.C. 6
Freilich et al (2005)
JMB ??
Extension and Evolution of Pathways:
The integration of the steroid biosynthesis pathway
into the sterol biosynthesis pathway
sterol
Universal
metazoa
human
cholesterol
bile acid
steroid hormone
Shiri Freilich
Enzyme Structure, Function and Evolution
Outstanding Research Questions:
– How is catalysis performed
• Principles of catalysis?
• E.C. numbers
– How do enzymes evolve?
– Can we predict enzyme function
from structure?
– Can we design new enzymes?
– What is the enzyme complement
in different organisms and how
does it evolve?
Need a list of active sites
Trypsin
What constitutes a catalytic residue?
• Direct involvement in the reaction mechanism
• Polarises or alters the pKa of a residue or water
molecule which is directly involved in the reaction
mechanism
• Polarises or activates part of the substrate
(e.g. making a bond more susceptible to cleavage)
• Stabilisation of a transition-state intermediate
http://www.ebi.ac.uk/thornton-srv/databases/CSA
The Catalytic Site Atlas: a resource of catalytic sites and residues identified in
enzymes using structural data.
Porter, Bartlett, & Thornton Nucl. Acids. Res. (2004) 32: D129-D133.
-lactamase Class A;
EC 3.5.2.6;
PDB: 1btl
– Reaction: -lactam + H2O  -amino acid
– Active site residues: S70, K73, S130, E16
N
N
O
O
HO
H
Ser
O
H
H
Ser
NH2
Lys
NH3+
N
H
O
O
Glu
O O
O
Ser
O
Ser
OH
Glu
Lys
N
H
HO
Ser
O
O
Ser O
Lys
HO
Ser
O
H
NH
O
NH3+
Lys
Ser
Comparison of CSA, SwissProt & PDB (2004)
Porter, Bartlett et al, 2004 NAR
Ligand Selectivity
Conformational Change
Templates
Metabolome
Binding Site Diversity
Spherical Harmonics
Catalytic Site Atlas
CSA Coverage and Annotations Generated
• Current entries in CSA from Literature = 737
• Current proteins in UniProt annotated by homology = 14,863
• Functional annotations by homology are more accurate if
catalytic residues are checked and conserved
(George et al (2005) PNAS)
BUT no possibility of storing or querying the
proposed chemical mechanisms (which must
be available to identify the catalytic residues in
the CSA)
The MACiE Database - a Research Project
http://www.ebi.ac.uk/thornton-srv/databases/MACiE
Mechanism, Annotation and
Classification in Enzymes
G. L. Holliday, G. J. Bartlett, Daniel Almonacid
P. Murray-Rust, J.M.Thornton J. B. O. Mitchell
(Holliday et al Bioinformatics 2005 21:4315)
Why develop MACiE?
• To understand more about catalysis
– To gather information on mechanisms
– To compare and contrast mechanisms in different
proteins
– To help validate enzyme mechanisms
– To study the evolution of mechanisms
– To develop mechanism-based classification of enzymes
– To help predict mechanism from structure
– To help design new enzymes
The MACiE Database
http://www.ebi.ac.uk/thornton-srv/databases/MACiE
Content in MACiE
●
●
Enzyme Name: fructose-bisphosphate aldolase
E.C. Classification: (EC 4.1.2.13)
–
●
EC 4.1.2.7
Reference Structure: PDB 1b57
–
–
–
–
–
●
Obsolete EC codes associated with entry:
Domain classification: CATH 3.20.20.70
UniProt code: P11604
Specie: Escherichia coli (Bacteria)
Cofactors: Zn2+ and Na+
Catalytic residues: Asp109, Glu182, Asn286
Links
Classifying Residue Catalytic Function
Hydrogen Donor, Hydrogen bond acceptor, Proton Relay
Nucleophile, Electrophile
Radical relay, Hydride relay
Radical Donor, Radical stabiliser
Leaving group , Steric role, Charge stabiliser
Covalently attached, Metal ligand
Overall Reaction
fructose-bisphosphate aldolase
O
H
O
O
O
P
O-
+
O-
H
O
O
O
H
O
O
O
O-
P
O
O-
O-
P
O-
O
O
O
O
H
glycerone phosphate +
D-glyceraldehyde
3-phosphate
O
P
O-
H
H
D-fructose 1,6-bisphosphate
O-
Step Annotation in MACiE
Step 1: Reactants
+
O Na
O
O
P
Glu182
O-
O-
O
O
H
O
H
H
H
O
O
Asp10 9
Zn2+
O
H N
H
Asn286
Step 1: Mechanism
Mechanism:
Proton transfer
Keto-enol tautomerisation (assisted)
+
O Na
O
O
O
O
-
O P
O-
H
Zn
H
H
O
O P
H
O
O
H N
H
O H
O
2+
O
H
O
O Na+
O
H
O
O H
O
O
-
O-
-
P
Glu182
OO P
O
O-
-
Glu1 82
Asn286
Asp10 9
Mechanism Components:
Overall substrate used
Intermediate Formed
Bond Formed = O-H
Bond Cleaved = C-H
Bond(s) changed in Order = C-C,1 to C=C, 2
C=O,2 to C-O, 1
O
O-
O-
O
Zn2+
H
H
O
H
O
H
O
Asp109
O
H N
H
Asn286
Step 1: Cofactors
Mechanism:
Proton transfer
Keto-enol tautomerisation (assisted)
OO P
O
+ Cofactor
O
O Na
O
O
O
O
-
O
O
-
O P
O-
H
Zn
H
2+
O
H
H
H N
H
O
O H
O
Cofactor
O
O
O Na+
O
H
O
O H
-
P
Glu182
O-
O P
H
O
-
Glu1 82
Asn286
Asp10 9
Mechanism Components:
Overall substrate used
Intermediate Formed
Bond Formed = O-H
Bond Cleaved = C-H
Bond(s) changed in Order = C-C,1 to C=C, 2
C=O,2 to C-O, 1
O
O-
O-
O
Zn2+
H
H
O
H
O
H
O
Asp109
O
H N
H
Asn286
Step 1: Spectator Residues
Mechanism:
Proton transfer
Keto-enol tautomerisation (assisted)
OO P
O
+ Cofactor
O
O Na
O
O
O
O
-
O
O
-
O P
O-
H
Zn
H
2+
O
H
H
O
Asn286
Spectator
Side Chain
Hydrogen Bond Acceptor
Hydrogen Bond Donor
Transition State Stabiliser
O H
O
Cofactor
O
O
O Na+
O
H
O
O H
-
P
Glu182
O-
O P
H
O
-
Glu1 82
H N
H
Asp10 9
Spectator
Side Chain
Mechanism Components:
Hydrogen Bond Acceptor
Overall substrate used
Intermediate Formed
Bond Formed = O-H
Bond Cleaved = C-H
Bond(s) changed in Order = C-C,1 to C=C, 2
C=O,2 to C-O, 1
O
O-
O-
O
Zn2+
H
H
O
H
O
H
O
Asp109
O
H N
H
Asn286
Step 1: Reactant Residues
Mechanism:
Proton transfer
Keto-enol tautomerisation (assisted)
OO P
O
+ Cofactor
O
Na
O
Reactant
Side Chain
Proton Acceptor
O
Glu182
O
-
O
O
-
O P
O-
H
Zn
H
2+
O
H
H
O
Asn286
Spectator
Side Chain
Hydrogen Bond Acceptor
Hydrogen Bond Donor
Transition State Stabiliser
O H
O
Cofactor
O
O
O Na+
O
H
O
O H
O-
P
O
O-
O P
H
O
-
Glu182
H N
H
Asp10 9
Spectator
Side Chain
Mechanism Components:
Hydrogen Bond Acceptor
Overall substrate used
Intermediate Formed
Bond Formed = O-H
Bond Cleaved = C-H
Bond(s) changed in Order = C-C,1 to C=C, 2
C=O,2 to C-O, 1
O-
O-
O
Zn2+
O H
H
O
H
O
H
O
Asp109
O
H N
H
Asn28 6
Mechanism:
Bimolecular Nucleophilic Addition
Proton Transfer
Aldol Addition
O-
Similarly Step 2
is annotated
O P
O-
O
O H
O Na+ Cofactor
Spectator
Side Chain
O
O P
H
OOZn2+ Cofactor
O
Glu182
O
H
H
O
H
O
Where the information is available
the rate determining step is annotated
Zn2+ Cofactor
O
H
O
H
O-
O
Asp109
H N
H
H
Asn286
H N Spectator
H Side Chain
Charge Stabiliser
Hydrogen Bond Donor
Steric Role
O
Rate Determining
Step
O
O H
O
Reactant
Side Chain Asp109
Proton Donor
Hydrogen Bond Acceptor
Mechanism Components:
Hydrogen Bond Donor
Overall substrate used
Overall product Formed
Intermediate Terminated
Bond Formed = C-C, O-H
Bond Cleaved = O-H
Bond(s) changed in Order = C=C, 2 to C-C, 1
C-O, 1 to C=O, 2
C=O, 2 to C-O, 1
Na+ Cofactor
Glu182
O-
Asn28 6
OO
P
O
O-
O
H
O P OO
OH
O
O
HH
O
H
+
O Na
P
O
O
Glu182
Step 3 is
annotated
O-
OO-
O
H
Zn2 +
O
H
H
H
O
O
O
H N
H
Asp109
Asn286
MACiE always endeavours to return the enzyme
to its ground state.
This is often inferred, which is noted in the annotation
Inferred Return
Step
OO
P
OO
O
Mechanism:
Proton transfer
Na+ Cofactor
O H
H
H
O
Glu182
Reactant
O H
Side Chain
Proton Donor H
O
Hydrogen Bond Donor
H
OO
Reactant
Side Chain Asp1 09
Proton Acceptor
Hydrogen Bond Acceptor
Zn2+ Cofactor
O
H N
H
Asn286
Spectator
Side Chain
Mechanism Components:
Proton Relay
Bond Formed = O-H
Bond Cleaved = O-H
Finally: any spontaneous
changes are included
OO
P
O O-
O
O
O P O-
H
-
O
O
H
O
HH
O
H
Occurs outside enzyme
O
O P O
O-
O
O
H
O
O P OO
OO H
H
These are often spontaneous and occur outside the enzyme
There is no other annotation involved in steps like this
Complete Reaction Annotation
Mechanism:
Proton transfer
Keto-enol tautomerisation (assisted)
OO P
O
+
OO Na Cofactor
Reactant
Side Chain
Proton Acceptor
O
Glu18 2
O-
Mechanism:
Bimolecular Nucleophilic Addition
Proton Transfer
- Aldol Addition
O
O
-
P
O
O
O-
O P
O-
H
Spectator
Side Chain
O
H
Zn2+ Cofactor
H
O
H
H
O
O H
O
O
O H
O Na+ Cofactor
O
H
O
Glu18 2
O H
Asn2 86
Spectator
Side Chain
Hydrogen Bond Acceptor
Hydrogen Bond Donor
Transition State Stabiliser
H N
H
H
H
O-
OOZn2 + Cofactor
O
O
P
O
O
H
O
Asp1 09
O
Spectator
Reactant
Side Chain
Side Chain Asp10 9
Mechanism Components:
Hydrogen Bond Acceptor
Proton Donor
Overall substrate used
Hydrogen Bond Acceptor
Intermediate Formed
Hydrogen Bond Donor
Bond Formed = O-H
Bond Cleaved = C-H
Bond(s) changed in Order = C-C,1 to C=C, 2
C=O,2 to C-O, 1
Inferred Return
Step
OO
OP
O
O
Mechanism:
Proton transfer
Na+ Cofactor
O H
H
H
O
Glu18 2
Reactant
O H
Side Chain
Proton Donor H
Hydrogen Bond Donor O
H
OO
Reactant
Side Chain Asp109
Proton Acceptor
Hydrogen Bond Acceptor
Asn2 86
H N
Spectator
H Side Chain
Charge Stabiliser
Hydrogen Bond Donor
Steric Role
Mechanism Components:
Overall substrate used
Overall product Formed
Intermediate Terminated
Bond Formed = C-C, O-H
Bond Cleaved = O-H
Bond(s) changed in Order = C=C, 2 to C-C, 1
C-O, 1 to C=O, 2
C=O, 2 to C-O, 1
Rate Determining
Step
Zn2+ Cofactor
O
H N
Asn286
H Spectator
Side Chain
Mechanism Components:
Proton Relay
Bond Formed = O-H
Bond Cleaved = O-H
OO
P
O
O-
O
O
O P O-
H
OO
H
O
HH
O
H
Occurs outside enzyme
O
O P O
O-
O
-
O
H
O
O P OO
OH
O
H
Searching MACiE
General Searches
●
●
●
●
Query MACiE by reaction comments
Query MACiE by enzyme and species
(scientific and common) names
Query the chemical changes in MACiE
Overall reactants and products (by KEGG and
ChEBI compound id or compound name)
Frequencies of amino acid reactants
performing a given function
Combining the amino acid and
functional clusterings
No reactant
Function
No strong preference
for function
Strong preference
for acid/base
function
Roles of catalytic residues and
mechanistic steps in homologous
enzymes of different function
– How do enzymes modify the chemical
reaction they catalyse, using the
same structural scaffold?
– Do catalytic residues conserve their
role and / or identity in enzymecatalysed reactions?
Gail Bartlett
Methods
178 enzyme dataset
with assigned catalytic
residues and proposed
mechanism
PSIBLAST run against NRDB
+ PDB (cutoff e=10-5)
Structurally equivalent
catalytic residues
Catalytic mechanisms
Twenty-seven pairs of
homologous proteins
of totally different
function (at primary
EC level)
Structural alignment performed
using SSM server
Information manually extracted
from literature
Comparison of
function, active
site, catalytic
residues and
catalytic
mechanism
Results - overview
• 27 pairs of proteins
• 3 enzyme / nonenzyme pairs
• 24 enzyme / enzyme pairs, from 21 enzyme
superfamilies
Change of function (EC) class
Function Y EC 1
EC 2
EC 3
EC 4
EC 5
EC 6
Nonenzyme
EC 1 (oxidoreductases) -
0
3
3
0
1
2
EC 2 (transferases)
-
2
1
1
0
0
-
3
1
0
1
-
7
1
0
-
0
1
-
0
Function X
EC 3 (hydrolases)
EC 4 (lyases)
EC 5 (isomerases)
EC 6 (ligases)
Enzyme / enzyme pairs
• All but one enzyme pair have their
active site located at the same place in
the protein fold
• Substrates and / or products shared by
11 pairs
• Cofactor shared by 5 enzyme pairs
Metal ion binding sites
Conserved
metal ion type
Altered metal
ion type
Conserved
metal ion
function
5
0
Altered metal
ion function
3
4
Twelve enzyme pairs
conserve metal binding sites
and ligands to the metal ions
are structurally aligned
Where the metal ion type has altered, subtle mutations
to the ligand binding site have occurred
Rubredoxin oxygen oxidoreductase /
metallo--lactamase
Rubredoxin oxygen
oxidoreductase reduces
dioxygen via a redox
cycle at a di-iron site
Fe ligand
Zn2+
ligand
His 79
His 82
Glu 81
His 84
Asp 83
Asp 86
His 146
His 145
Asp 165
Cys 164
His 226
His 206
Metallo--lactamase uses a
Zn2+-activated hydroxyl for
nucleophilic attack on the lactam substrate
Catalytic residue pairs
Catalytic residue pairs
35
Unconserved residue
identity
Conserved residue
identity
Number of pairs
30
25
20
15
10
5
0
Identical role
Different role
Role of residue
Role in one protein only
Change in residue identity and
residue function
DNA hydrolysis
Xylose  Xylulose
Endonuclease IV / Xylose isomerase
Change in residue identity and
residue function
His 109
Trp 136
Base catalysed
ring opening,
followed by
intramolecular
hydride transfer
and ring closure
Zn2+-assisted
hydroxyl
performs
nucleophilic
attack on DNA
backbone
Endonuclease IV
Xylose isomerase
Evolution of Mechanism
7
7
2
8
Mechanisms share a common step at the beginning
of the overall reaction pathway, catalysed by
residues which are structurally equivalent in both
enzymes
Mechanisms share a common step somewhere in
the middle of the overall reaction path, catalysed by
residues which are structurally equivalent in both
enzymes
Mechanisms share common steps at the beginning
and end of the overall reaction path, catalysed by
residues which are structurally equivalent in both
enzymes, but have a different step in the middle
Mechanisms do not share any common steps
catalysed by structurally equivalent residues
Bartlett et al JMB
Common first step
dehydroquinate synthase / glycerol dehydrogenase
a. Dehydroquinate synthase
NAD+
HO
O2-C
HO H
H
H
O H
O
O
Zn
2+
H
OPO3
several stages
O2-C
HO
HO
OH
O
b. Glycerol dehydrogenase
H
HO
HO
O H
H
NAD+
O
Zn
2+
HO
H
O
HO
Common first step
dehydroquinate synthase / glycerol dehydrogenase
H287
E194
H275
dehydroquinate
synthase
H271
H269
Superposition of
Zn2+ and ligands
D169
H255
H252
glycerol
dehydrogenase
Conclusions
• Enzymes are economical in their use of active
site residues and features
• It is more likely for residues conserving
function to also conserve their identity
• Residues not conserving function tend to
mutate
• Tend to find common mechanistic steps at the
beginning and ‘middle’ of reaction paths –
possibly the most energetically difficult step or
intermediate is conserved
Future
• Increase coverage in MACiE
• Analyse Catalytic Mechanisms
– Ingold Reaction Types; effects of non-polar
residues
• Evolution of enzymes, pathways &
metabolism in different organisms & tissues
• Design??
Acknowledgements
•
Gail Bartlett, Craig Porter, Jonathan Barker
James Torrance
Alex Gutteridge
Gemma Holliday
• The MACiE Team – Cambridge Univ Chemistry Dept
John Mitchell, Daniel Almonacid, Peter Murray-Rust
• BBSRC, MRC, Wellcome Trust, EMBL