The Chemistry of Protein The Chemistry of Protein CatalysisCatalysis

John Mitchell

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 (EBI), Peter Murray-Rust, Gail Bartlett (EBI),

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

Only deals with overall reaction

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

Coverage of MACiE

Representative – based on a non-homologous dataset,and chosen to represent each available EC sub-subclass.

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.-.-.-

56 EC 1.2.-.-

184 EC 1.2.3.-

1312 EC 1.2.3.4

MACiE covers:

6 EC 1.-.-.-

53 EC 1.2.-.-

156 EC 1.2.3.-

199 EC 1.2.3.4

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., accepted (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

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 FunctionEvolution of Enzyme Function

D.E. Almonacid et al., to be published

EC is our Functional Classification

Enzyme Commission (EC) Nomenclature, 1992, Academic Press, San Diego, 6th Edition

Chemical reaction

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.

Work with domains - evolutionary & structural units of proteins.

Map enzyme catalytic mechanisms to domains to quantify convergent and divergent functional evolution of enzymes.

Domains

CATH is our Structural Classification

Orengo, C. A., et al. Structure, 1997, 5, 1093

Numbers of CATH code occurrences per EC number

C

A

T

H

c.-.-.- c.s.-.- c.s.ss.- c.s.ss.sn

3.17

11.00

28.00

38.33

1.73

3.27

4.89

5.80

1.38

1.93

2.24

2.46

1.11

1.60

1.19

1.22

Results: Convergent Evolution

2.46 CATH/EC reaction

Convergent Evolution

Numbers of CATH code occurrences per EC number

C

A

T

H

c.-.-.- c.s.-.- c.s.ss.- c.s.ss.sn

3.17

11.00

28.00

38.33

1.73

3.27

4.89

5.80

1.38

1.93

2.24

2.46

1.11

1.60

1.19

1.22

Results: Convergent Evolution

2.46 CATH/EC reaction: Convergent EvolutionAn average reaction has evolved independently in 2.46 superfamilies

EC reactions/CATH

C4.75

19.50

39.25

90.00

c.-.-.-c.-.-.-

c.s.-.-c.s.-.-

c.s.ss.-

c.s.ss.sn

A3.14

7.00

10.48

17.90

T1.36

1.79

2.08

3.05

H1.20

1.36

1.462.05

database entries/CATH

2.18

Results: Divergent Evolution

1.46 EC reactions/CATH Divergent Evolution

EC reactions/CATH

C4.75

19.50

39.25

90.00

c.-.-.-c.-.-.-

c.s.-.-c.s.-.-

c.s.ss.-

c.s.ss.sn

A3.14

7.00

10.48

17.90

T1.36

1.79

2.08

3.05

H1.20

1.36

1.462.05

database entries/CATH

2.18

Results: Divergent Evolution

1.46 EC reactions/CATH: Divergent EvolutionAn average superfamily has evolved 1.46 different reactions

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

45

Energy Landscape

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

Dr Gemma Holliday

Dr Daniel Almonacid

Dr Noel O’Boyle

Prof. Janet Thornton (EBI)

Dr Peter Murray-Rust

Dr Florian Nigsch

ACKNOWLEDGEMENTSACKNOWLEDGEMENTS

Cambridge Overseas

Trust