Daniel E. Almonacid, Gemma L. Holliday, Gail J. Bartlett, Noel M. O’Boyle, Peter Murray-Rust, Janet M. Thornton and John B. O. Mitchell Enzyme Mechanism Annotation and Classification

Enzyme Nomenclature and Classification EC Classification Class Subclass Sub-subclass Serial number

 In 1965, the first three-dimensional structure for an enzyme was reported. Enzyme Nomenclature and Classification β-lactamases EC  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

MACiE Mechanism, Annotation and Classification in Enzymes. MACiE database

Unimolecular Heterolytic Bimolecular Intramolecular Elimination Unimolecular HomolyticBimolecular Intramolecular ElectrophilicBimolecular Intramolecular Addition NucleophilicBimolecular Intramolecular HomolyticBimolecular Intramolecular Unimolecular ElectrophilicBimolecular Intramolecular Unimolecular SubstitutionNucleophilicBimolecular Intramolecular Unimolecular HomolyticBimolecular Intramolecular Ingold, C. K. Cornell University Press, Repertoire of enzyme catalysis

Enzyme chemistry is largely nucleophilic

“Other reactions” and Named organic reactions currently supported in MACiE ______________________________________________ Aldol CondensationHydride Transfer Amadori RearrangementIsomerisation A-S N 1Michael Addition A-S N 2Nucleophilic Attack A-S N iPericyclic Reaction Claisen RearrangementProton Transfer CondensationRadical Formation E1cbRadical Propagation Group TransferRadical Termination HeterolysisRedox HomolysisTautomerisation ______________________________________________ Repertoire of enzyme catalysis

Proton transfers Ad N 2E1SN2SN2E2 Radical reactions Tautom.Others Repertoire 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 residues

CATH – EC Relationships Numbers of CATH code occurrences per (partial) EC number C A T Dataset of function-domain pairs from PDB H c c.s.-.- c.s.ss.- c.s.ss.sn Putative evolutionary units of protein structure (H) and units of enzyme function (c.s.ss.-)

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) 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!

CONCLUSIONS  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  TyMeS: Types of Mechanism and Structure.

ACKNOWLEDGEMENTS Cambridge Overseas Trust

  MACiE database QUESTIONS? MACi E Mechanism, Annotation and Classification in Enzymes.

Functionality for amino acids currently supported in the MACiE ________________________________________________ Activating residueProton acceptor Charge destabiliserProton donor Charge stabiliserProton relay Covalently attachedRadical acceptor ElectrophileRadical donor Hydride relayRadical relay Hydrogen bond acceptorRadical stabiliser Hydrogen bond donorSpectator Leaving groupSteric hindrance Metal ligandUnknown function NucleophileUnspecified steric role ________________________________________________ Function of catalytic residues

Catalytic propensity % 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 residues