Proteases / Proteolysis
• – Proteases or peptidases are
enzymes that catalyze the break
down of peptide bonds in
• – Proteolysis is the process of
controlled or uncontrolled
degradation of proteins/peptides.
Diabetes type 2
Diabetes & obesity
• Enzyme and substrate become linked in a covalent
bond at one or more points in the reaction pathway
• The formation of the covalent bond provides
chemistry that speeds the reaction
• Serine proteases also employ general acid-base
Trypsin, chymotrypsin, elastase, thrombin (blood clotting
cascade), subtilisin (bacterial protease), plasmin (degrades
fibrin polymers of blood clots), TPA (cleaves plasminogen),
• All involve a serine in catalysis - thus the name
• Ser is part of a "catalytic triad" of Ser, His, Asp
• Serine proteases are homologous, but sequence locations
of the three crucial residues differ somewhat
– Enzymologists agree, however, to number them always
as His57, Asp102, Ser195
The Serine Proteases
The amino acid
trypsin, and elastase.
• similar structures, yet
Proteases: Members of the same family are usually evolutionarily related
Elastase, Thrombin, Subtilisin,
Factor D, Factor B, etc.
Gastricsin, Cathepsin D, Renin,
HIV protease, etc.
Matrix Metalloprotease (MMP)
Papain, Calpain, Cruzipain,
Catalytic Residues of Serine Proteases
The only amino acid with a primary hydroxyl,
often seen as a nucleophile in enzyme active sites.
Can act as a hydrogen bond donor or acceptor.
Histidine is the only amino acid with a pKa in the
physiological range, hence often seen in active
sites when donation or abstraction of a proton
is needed. The protonated form shown has a pKa
around 6 for the indicated proton , meaning it's
mainly neutral at pH 7.
Side chain pKa is about 4.
One of two negatively charged amino acids.
Did chymotrypsin evolve via gene
The 2 domains have similar structure
but no sequence identity
Single domains do have limited activity
Fact that active site loops comprise is
clustered in same two loops supports
• Chymotrypsin structure has
two anti-parallel beta-barrel
• Active site is formed by 2
loop regions from each
Serine Protease Mechanism
A mixture of covalent and general acid-base catalysis
• Asp102 functions only to orient His57
• His57 acts as a general acid and base
• Ser195 forms a covalent bond with peptide to be
• Covalent bond formation turns a trigonal carbon into
a tetrahedral carbon
• The tetrahedral oxyanion intermediate is stabilized by
N-Hs of Gly193 and Ser195
4 Important Features:
1. Base (H) to accept proton from
S-OH to facilitate tetrahedral
2. Tight binding of tetrahedral
transition state accomplished via
oxyanion hole--H+ also stableizes
negatively charged transition state
3. Nonspecific binding (due to
main chain atoms H-bonding to
main chain atoms of loop region in
enzyme) also contributes to
affinity/stabilization of transition
4. Specificity pocket
His57 acts as
First reaction step after substrate binding
The “intermediate” is highly unstable.
It is a close mimic of the transition state.
His57 now acts
as a general acid
Oxyanion hole Stabilizes the
negative charge on the acyl
This covalent intermediate is stable and
can be isolated. However, it has to be
Resolved to generate free enzyme and
A water molecule jumps in
His57 functions again
as a general base
Oxyanion Hole functions
similar as in previous steps.
…and again His57 as a
Again, this is not a stable
or isolable intermediate
Assembly, Budding and Maturation of HIV-1
• First drug developed for HIV-1 was AZT which inhibits Reverse Transcriptase (RT)
• AZT is a nucleoside analog that on conversion to triphosphate in the cell, inhibits RT.
• Unfortunately RT inhibitors only slow progression of HIV infection but do not stop it.
1. AZT-like drugs are toxic to bone marrow cells so can only taken as small doses.
2. RT unlikie other DNA polymerases cannot repair its mistakes so mutations
eventually make the protein resistant to RT inhibitors.
HIV -1 protease excises itself from gag-pol then cleaves other proteins on
gag and gag-pol to produce its pathogenic mature form.
If HIV-1 protease is inactivated either mutagenically or via inhibiotr, the
virion remains noninfectious.
The Aspartic Proteases
Pepsin, chymosin, cathepsin D, renin and
All involve two Asp residues at the active site
These two Asp residues work together as
general acid-base catalysts
Most aspartic proteases have a tertiary
structure consisting of two lobes (N-terminal
and C-terminal) with approximate two-fold
HIV-1 protease is a homodimer
The Aspartic Proteases
Most aspartic proteases exhibit a
two-lobed structure. Each lobe
contributes one catalytic aspartate
to the active site. HIV-1 protease
is a homodimeric enzyme, with
each subunit contributing a
catalytic Asp residue.
Structures of (a) HIV-1
protease, a dimer, and (b)
pepsin, a monomer. Pepsin’s Nterminal half is shown in red; the
C-terminal half is shown in blue.
Protease Inhibitors Block the Active Site of HIV-1
HIV-1 protease complexed with the inhibitor Crixivan (red)
made by Merck. The “flaps” that cover the active site are
green; the catalytic active site Asp residues are violet.
Protease Inhibitors Give Life to AIDS Patients
Protease inhibitors as AIDS drugs
If the HIV-1 protease can be selectively inhibited,
then new HIV particles cannot form
Several novel protease inhibitors are currently
marketed as AIDS drugs
Many such inhibitors work in a culture dish
However, a successful drug must be able to kill the
virus in a human subject without blocking other
essential proteases in the body