Enzyme structure, specificity and mechanism

substrate binding - lock and key model

complimentary geometry and interactions between the enzyme and substrate(s) are needed for binding

substrate binding - induced fit model

enzymes are flexible proteins and able to change conformation, substrate binding often alters the shape of the enzyme by using the binding energy, optimal interactions with the enzyme occur only with the transition state.

Induced fit maximises the binding interactions with the transition state to reduce the activation energy required for the reaction to be accelerated

substrate binding - non-covalent interactions

substrate binding interactions 

antibiotic resistance to chloramphenicol is often the result of enzyme catalysed acetylation of the antibiotic which stops it from binding its target 

serine proteases

digestive enzymes which hydrolyse peptide bonds to break down proteins and peptides

how do different serine proteases have different substrate selectivity 

substrate binding pocket site close to the active site dictates selectivity 

serine proteases - essential amino acids for catalysis

Biochemical identification of the essential catalytic serine was achieved using an organophosphorus compound to chemically label active site group.

Identifying essential amino acids in the structure and mechanism through sequence alignments

% identity - same amino acid

% similarity - amino acid with similar properties

serine proteases - catalytic mechanism

Catalytic triad: catalytic residues of Ser195, His57 and Asp102 form part of the active site pocket and are conserved across serine proteases.

3 major challenges

1. Stable peptide bond due to the resonance structure

2. Water is a poor nucleophile

3. The amine product is a poor leaving group

stabilisation of transition state

For serine proteases the tetrahedral oxyanion intermediate and transition state is stabilised through forming hydrogen bonds to the enzyme (oxyanion hole)

4 important concepts in serine protease catalysis

1. Catalytic triad increases the reactivity of the catalytic serine

2. The oxyanionic hole stabilises the intermediates and transition states

3. Covalent catalysis provides an effective mechanism to reduce the activation energy for catalysis

4. The covalent enzyme intermediates are hydrolysed to reform the active enzyme

preorganised active site electrostatics

Active site electrostatics preferentially stabilise the transition state changes which form transiently during the reaction.

general acid/base catalysis

Uses ionisable amino acids to provide or accept a proton as part pf the reaction cycle to accelerate catalysis

HAD (haloacid dehalogenases) phosphatases (hydrolases)

Magnesium (Mg2+) dependent enzymes

metal ion catalysis 

Metalloenzymes - contain tightly bound metal ions. Fe2+/3+, Cu2+, Co3+, Zn2+

Metal-activated enzymes - loosely bound metal ions from solution often with the substrate, Mg2+, Ca2+, K+, Na+

1. Bind substrates and help orientate for catalysis

2. Mediate oxidation-reduction reactions

3. Electrostatic stabilisation and shield negative charge (acid/base)

4. Polarise bonds coordinating to the metal ion

Metallo-beta-lactamases are used by antibiotic resistant pathogenic bacteria to break down beta-lactam antibiotics

cytochrome p450 metalloenzymes

metal cofactors mediate oxidation-reduction reactions