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

jbom1@cam.ac.uk

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