Lecture 1: Enzyme kinetics (Michaelis Menten)
Case study – chymotrypsin
Kinetics of multi-substrate reactions
Lecture 2: Enzyme mechanisms
Inverting and retaining glycosidases
Case study – lysozyme
Hydroxylase enzymes – use of cofactors
Case study 2 – phenylalanine hydroxylase (including
pre-steady state kinetics)
Lecture 3: Enzyme inhibition
Classes of enzyme inhibitor and mechanisms
Effects on enzyme kinetics
Use of kinetic data to identify potent inhibitors.
Lecture 4: Protease Inhibition
Case study – HIV aspartic protease
Mechanism and inhibitor development
Other proteases
Enzymology: Lecture 4
PROTEASES: Enzymes that catalyse the hydrolytic cleavage of peptide bonds
Enzymology: Lecture 4
4 MECHANISTIC CLASSES: • Serine proteases - Neitzel, J. J. (2010) Enzyme Catalysis: The Serine
Proteases . Nature Education 3(9):21
• Threonine proteases • Cysteine proteases • Aspartyl proteases • Metalloproteases
Proteases
THE CATALYTIC TRIAD:
Enzymology: Lecture 4
Asp-His-Ser Variations in the triad are possible, while still maintaining the chemical properties to enable hydrolysis Dodson and Wlodawer (1998) Trends in Bioch Sci, 23: 347. Buller and Townsend (2013) Proc Natl Acad Sci, 110: E653
Acid/base - nucleophile
Proteases
Amino acid side chains (and the peptide backbone) provide a repertoire
of functional groups for catalysis and binding
Enzymology Lecture 1 How Enzymes Promote Catalysis
THREONINE PROTEASES:
Enzymology: Lecture 4
Proteasomal degradation machinery
• N-terminal nucleophile hydrolase (Ntn favourable due to steric hindrance) • OH group on threonine side chain • Enzyme-bound H2O acts as as general base to activate nucleophile • Ekici OD et al, Protein Sci, 2008, 17: 2023.
Proteases
CYSTEINE PROTEASES:
Enzymology: Lecture 4
• catalytic diad or triad (aspartate) • His is proton-withdrawing to promote nucleophilic attack • Common throughout biological processes, e.g. papain
Cysteine Histidine
Peptide bond Tetrahedral intermediate
Thioester intermediate Hydrolysis
Proteases
METALLO PROTEASES:
Enzymology: Lecture 4
• most commonly Zn, tetrahedral conformation • Endopeptidases, e.g. tetanus and botulism neurotoxins, or exopeptidases, e.g. carboxypeptidase • Coordinated by three residues, usually charged • Hydrolytic H2O also coordinates metal.
H2O is induced to act as a nucleophile
ANTIBIOTIC RESISTANCE Metallo-β-Lactamases:
Proteases
Enzymology: Lecture 4
Metallo-β-Lactamases
Di-Zn Mechanism:
Mono-Zn Mechanism:
Karsisiotis et al (2014) Metallomics 6: 1181.
Proteases
Enzymology: Lecture 4
ANTIBIOTIC RESISTANCE Metallo-β-Lactamases, NDMs:
Proteases
ASPARTATE PROTEASES:
Enzymology: Lecture 4
• Aspartate residue induces nucleophilic attack by H2O
One aspartate is deprotonated pH rate profile
Nucleophilic H2O attacks carbonyl C to generate tetrahedral oxyanion intermediate; stabilised by alternative Asp
Tetrahedral collapse to yield hydrolysis products
General acid-base mechanism precludes need for metal or covalent intermediate
Proteases
Enzymology: Lecture 4
www.aids.gov
HIV Aspartate Protease
Enzymology: Lecture 4
www.aids.gov
HIV Aspartate Protease
Enzymology: Lecture 4
HIV aspartate protease cleaves precursor proteins into active proteins required for viral core, viral coat, reverse transcriptase, ribonuclease and viral replication Major target for anti-HIV drugs
HIV Aspartate Protease
HIV ASPARTATE PROTEASE
Enzymology: Lecture 4
Homodimeric aspartyl protease – chains are identical, not covalently bound, symmetric association
Active site at base of dimer interface
Aspartate residues from each monomer form active site
‘β-hairpin flaps’ enclose substrate, 7Å movement on
binding
Anderson et al (2009) Handbook Exp Pharmacol 189: 85
Contrast to mammalian aspartate proteases – two active sites in one protein
HIV Aspartate Protease
Enzymology: Lecture 4
pKa = 3.1
pKa = 5.2
Crystallographic evidence for tetrahedral intermediate Kovalevsky et al (2007) Biochemistry 46: 14854
Confirmation of HIV Protease as an Aspartate Protease:
Structural Studies
HIV Aspartate Protease
Confirmation of HIV Protease as an Aspartate Protease:
Site directed mutagenesis of Asp-25 to Asn, Thr or Ala
Result: Total loss of proteolytic activity
Enzymology: Lecture 4
Pepstatin inhibition
Result: Ki in nM range – standard inhibitor of aspartate proteases, inhibited HIV protease
pH profile indicates Asp with elevated pKa
HIV Aspartate Protease
Enzymology: Lecture 4
HIV ASPARTATE PROTEASE: URGENT NEED FOR INHIBITORS
Crystal structure of HIV aspartate protease search for specific inhibitors Protease sites in gag and gag-pol proteins not well conserved , but 3 of 8 sites not targets for human aspartate protease: PHE-PRO TYR-PRO Basis of transition state analogues:
HIV Aspartate Protease Inhibitors
Enzymology: Lecture 4 HIV Aspartate Protease Inhibitors
Enzymology: Lecture 4
HIV ASPARTATE PROTEASE: SAQUINAVIR
First FDA approved HIV protease inhibitor
Saquinovir binds in extended conformation, H bonding with the enzyme Carboxylate O interacts with flap Wlodaver and Vondrasek (1998) Annu Rev Biophys Biomol Struct 27: 249
HIV Aspartate Protease Inhibitors
Enzymology: Lecture 4
HIV THERAPY: STATE OF THE ART
• HIV protease inhibitors clearly prolong the lives of HIV-positive and AIDS sufferers
• All HIV-1 protease inhibitors initially cause a rapid and profound decline in plasma HIV load and immune system recovery
• Mutant forms of the protease will arise
• Combination therapies - protease inhibitor + inhibitors of other viral processes
• Plasma virus’ become undetectable, but not a cure; anti-HIV medications must be taken for life.
• Bioavailability issues and side effects
• Complicated molecules therefore expensive
HIV Aspartate Protease Inhibitors