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Daniel E. Almonacid, Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Gemma L. Holliday, Gail J. Bartlett, Noel M. O’Boyle, Peter Murray-Rust, Janet M. Thornton Noel M. O’Boyle, Peter Murray-Rust, Janet M. Thornton
and John B. O. Mitchelland John B. O. Mitchell
Enzyme Mechanism Annotation and Classification
Enzyme Nomenclature and Enzyme Nomenclature and ClassificationClassificationEC ClassificationEC Classification
Class
Subclass
Sub-subclass
Serial number
In 1965, the first three-dimensional structure for an enzyme was reported.
Enzyme Nomenclature and Enzyme Nomenclature and ClassificationClassification
OHH
NO
R R'
R''NH
O O R''
R R'
β-lactamases EC 3.5.2.6
Advances on biochemical techniques, e.g. site directed mutagenesis. We know the function of amino acids in the chemical reaction mechanisms of enzymes.
There is a need to develop new classification schemes
MMechanism, AAnnotation and CClassification iin EEnzymes.http://www-mitchell.ch.cam.ac.uk/macie/
MACiE databaseMACiE database
UnimolecularHeterolytic Bimolecular
IntramolecularElimination
UnimolecularHomolytic Bimolecular
Intramolecular
Electrophilic BimolecularIntramolecular
Addition Nucleophilic BimolecularIntramolecular
Homolytic BimolecularIntramolecular
UnimolecularElectrophilic Bimolecular
Intramolecular
UnimolecularSubstitution Nucleophilic Bimolecular
Intramolecular
UnimolecularHomolytic Bimolecular
Intramolecular
Ingold, C. K. Cornell University Press,
1969.
Repertoire of enzyme catalysisRepertoire of enzyme catalysis
Repertoire of enzyme catalysisRepertoire of enzyme catalysis
0
20
40
60
80
100
120
140
HeterolyticElimination
HomolyticElimination
ElectrophilicAddition
NucleophilicAddition
HomolyticAddition
ElectrophilicSubstitution
NucleophilicSubstitution
HomolyticSubstitution
Reaction Types
Num
ber
of
steps
in M
ACiE
Intramolecular
Bimolecular
Unimolecular
Enzyme chemistry is largely nucleophilic
“Other reactions” and Named organic reactions currently supported in MACiE
______________________________________________
Aldol Condensation Hydride Transfer Amadori Rearrangement Isomerisation A-SN1 Michael Addition A-SN2 Nucleophilic Attack A-SNi Pericyclic Reaction Claisen Rearrangement Proton Transfer Condensation Radical Formation E1cb Radical Propagation Group Transfer Radical Termination Heterolysis Redox Homolysis Tautomerisation______________________________________________
Repertoire of enzyme catalysisRepertoire of enzyme catalysis
0
50
100
150
200
250
300
350
400
450
Reaction Types
Num
ber
of
ste
ps in M
ACiE
Protontransfers
AdN2 E1 SN2 E2 Radicalreactions
Tautom. Others
Repertoire of enzyme catalysisRepertoire of enzyme catalysis
In MACiE catalytic residues are those:
i) directly involved in the catalytic mechanism;
ii) modifying the pKa of a residue or water molecule directly involved in the catalytic mechanism;
iii) stabilising a transition state or intermediate;
iv) activating the substrate.
Bartlett, G. J. et al. J. Mol. Biol., 2002, 324, 105.
Porter, C. T. et al. Nucleic Acids Res., 2004, 32, D129.
Function of catalytic residuesFunction of catalytic residues
Function of catalytic residuesFunction of catalytic residues
0
2
4
6
8
10
12
14
Gly Ala Val Leu Ile Phe Pro Met Trp Ser Thr Cys Tyr Asn Gln Asp Glu Lys Arg His
Amino acids
Cata
lyti
c pro
pensi
ties
Main-chain spectatorSide-chain spectatorMain-chain reactantSide-chain reactant
CATH – EC RelationshipsCATH – EC Relationships Numbers of CATH code occurrences per (partial) EC number
C
A
T
Dataset of 31367 function-domain pairs from PDB
H
c.-.-.- c.s.-.- c.s.ss.- c.s.ss.sn
3.67
19.3
116
163
2.62
7.51
16.9
21.1
2.01
4.18
6.72
7.78
1.35
1.77
2.02
2.13
Putative evolutionary units of protein structure (H) and units of enzyme function (c.s.ss.-)
Enzyme evolution (1)Enzyme evolution (1)
• 7.78 distinct homologous superfamilies (CATH) per distinct function (c.s.ss.-)
• Average enzymes comprises 1.54 CATH homologous superfamilies
• 7.78/1.54 = 5.05
• Naively implies that each function has evolved approximately five times
• But ...
Enzyme evolution (2)Enzyme evolution (2)
• George et al. (Bioinf. 20, i130, 2004) did the same thing for SCOP vs. EC
• They found 3.5 SCOP superfamilies per EC sub-subclass (c.s.ss.-)
• Implies each function has evolved approximately twice
• Answer is highly method-dependent!
CONCLUSIONSCONCLUSIONS Enzyme catalysis exploits a limited area of chemical space. However, they catalyse almost all the reactions in the metabolism of all organisms.
Amino acids with alkyl or aryl side-chains act mainly as main-chain spectators. Amino acids with chemical functionality in their side-chains are divided into two groups, depending on whether or not they can also exploit their main-chain functionality.
TyMeSTyMeS: TyTypes of MeMechanism and SStructure.
ACKNOWLEDGEMENTSACKNOWLEDGEMENTS
Cambridge Overseas
Trust
MACiE database
QUESTIONS?QUESTIONS?
MMechanism, AAnnotation and CClassification iin EEnzymes.
http://www-mitchell.ch.cam.ac.uk/macie/
Functionality for amino acids currently supported in the MACiE
________________________________________________
Activating residue Proton acceptor Charge destabiliser Proton donor Charge stabiliser Proton relay Covalently attached Radical acceptor Electrophile Radical donor Hydride relay Radical relay Hydrogen bond acceptor Radical stabiliser Hydrogen bond donor Spectator Leaving group Steric hindrance Metal ligand Unknown function Nucleophile Unspecified steric role________________________________________________
Function of catalytic residuesFunction of catalytic residues
Catalyticpropensity
% Occurrence as a catalytic residue
% Total occurrence in the dataset=
Number of times occurs as a catalytic residue
Total number of catalytic residues
% Occurrence as a catalytic residue =
Number of times residue occurs in all enzyme sequences
Total number of residues in all enzyme sequences
% Total occurrence in the dataset =
Function of catalytic residuesFunction of catalytic residues
