second year enzymology lecture 4

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


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:


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:


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:


• 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:


• 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


Metallo-β-Lactamases

Di-Zn Mechanism:

Mono-Zn Mechanism:

Karsisiotis et al (2014) Metallomics 6: 1181.

Proteases


ANTIBIOTIC RESISTANCE Metallo-β-Lactamases, NDMs:

Proteases

ASPARTATE PROTEASES:


• 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


www.aids.gov

HIV Aspartate Protease


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


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



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


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


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: 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


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 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


second year enzymology lecture 4

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