Transcript Chymotrypsin

PROTEIN PHYSICS
LECTURE 24
PROTEINS AT ACTION:
BIND  TRANSFORM  RELEASE
BIND: repressors
- turn - 
Znfingers
DNA & RNA
BINDING
Leu-zipper
BIND  TRANSFORM  BIND: Repressors
-BINDING-INDUCED DEFORMATION
MAKES REPRESSOR ACTIVE, and IT BINDS TO DNA
BIND: Immunoglobulins
Immunoglobulin
BIND  TRANSFORM  RELEASE:
ENZYMES
chymotrypsin
Note small
active site
Chymotrypsin catalyses hydrolysis of a peptide
Spontaneous hydrolysis: very slow
Chymotrypsin
Chymotrypsin is one of the serine proteases.
Chymotrypsin is selective for peptide bonds with
aromatic or large hydrophobic side chains, such as
Tyr, Trp, Phe and Met, which are on the carboxyl side
of this bond. It can also catalyze the hydrolysis of
easter bond.
The main catalytic driving force for Chymotrypsin is
the set of three amino acid known as catalytic triad.
This catalytic pocket is found in the whole serine
protease family.
Properties of an Active Site
A shape that fits a specific substrate or
substrates only
Side chains that attract the enzyme
particular substrate
Side chains specifically positioned to
speed the reaction
The Catalytic Triad
chymotrypsin
CHAIN CUT-INDUCED DEFORMATION
MAKES ENZYME ACTIVE

active
cat. site
Chymotripsin
nonactive
cat. site
Chymotripsinogen
SER-protease:
catalysis
Stage #1
Chymotrypsin Protein
Hydrolysis
Chymotrypsin Protein
Hydrolysis
Stage #2
Chymotrypsin Protein
Hydrolysis
Stage #3
Chymotrypsin Protein
Hydrolysis
Stage #4
Chymotrypsin Protein
Hydrolysis
Stage #5
Chymotrypsin Protein
Hydrolysis
Stage #6
Transition State
Stabilization
Chymotrypsin Kinetics
The initial "burst" in
chymotrypsin-catalysed
hydrolysis of the p-nitrophenyl
acetate
CHYMOTRYPSIN ACTIVE SITE with INHIBITOR
Catalytic antibodies
ABZYM = AntyBody enZYM
Transition state (TS)
Antibodies
are
selected
to TS-like
molecule
Preferential
binding
of TS:
RIGID
enzyme
A novel approach to drug
delivery:
abzyme-mediated
drug activation
Levi Blazer
11/19/04
Immunology Review
Immunoglobulin G
Monoclonal vs.
polyclonal
http://www.path.cam.ac.uk/~mrc7/igs/img09.jpeg
Monoclonal antibody production
Why monoclonal?
Don’t tell PETA
Two forms of
hybridoma preps:
– Mouse Ascites
– In vitro tissue culture.
http://ntri.tamuk.edu/monoclonal/mabcartoon.gif
Abzymes
Catalytic monoclonal antibodies: usually IgG, although in theory all Ig
subclasses could be created.
Created by immunizing an animal against a transition state analog (TSA) of
the desired reaction.
Any non-lethal TSA antigen that can be coupled to a carrier protein can
potentially create a useful abzyme.
Energy
Abzyme stabilization of
transition state
ΔG
Progress
Abzymes – nearly endless
possibilities
Acyl-transfer
Cationic cyclization
Disfavored ring
closure
Aldol/Michaels
Hydride transfer
Oxy-cope
rearrangements
Abzymes
Specific for a
particular reaction
But - varied enough
to accept a variety of
substrates
Can be produced for
any non-lethal
antigen.
Easier to humanize
Reactive immunization
A novel method to select and create the
most catalytically active abzymes.
Use an immunogen that will react a
physiological pH or will bind covalently to a
B-cell receptor.
Reactive Immunization
Enaminone absorbs
at 316 nm
Overview of the old system
ADEPT – Antibody directed
enzyme prodrug therapy
Chemically modify a chemotherapy
agent to make it minimally toxic.
Prepare an antibody-enzyme
conjugate that catalyzes the
activation reaction
Use a localized injection of
conjugate to selectively activate
drug in tumor tissue.
Tumor
Suppression
Chemotherapy agent
Inactivator:
Removable through
enzyme/abzyme catalysis
Benefits of ADEPT
Minimized toxicity = better!
Localized activation
Potentially lower required doses
Problems with ADEPT
Immunological response to non-host
enzyme (the antibody section can be humanized)
Conservation of active sites across
species
Selectivity of enzyme
Hard to engineer
Mother Nature:
Better than Reingold
Enzymes catalyze many reactions faster
and with more specificity than synthetic
catalysts.
Problem: difficult to engineer an enzyme if
there is no natural analog.
Why not let Mother Nature do the design
work for us? -- ABZYME! --
Problems?
Antibodies bind molecules.
How can you use this in humans?
– Immune response
– Diffusion
– Protein stability
– Side reactions?
– Natural activation?
Cost!
Ethical concerns.
Methodology:
synthesize prodrug with
standard inactivator
Y
Administer prodrug and
catalytic antibody conjugate
separately.
Administer catalytic
antibody directly into
Tumor.
Normal Tissue
Localized activation
reduces unwanted toxicity
Tumor
Doxorubicin activation
Topoisomerase I & II inhibitor
By abzyme 38C2
Prodox synthesis
Conclusions
Abzyme conjugated ADEPT:
– Potentially more effective
– Less toxic for non-cancerous cells
– Sustainable for long periods of time due to
antibody half-life.