Download Proteases: Hydrolysis of Peptide Bonds

C. Walsh
Proteases: Hydrolysis of Peptide Bonds: Specificity and Mechanism
Proteases (protein hydrolases) catalyze amide (peptide) bond hydrolysis in protein or
peptide substrates:
Cellular and physiologic functions:
x Hydrolysis of peptide bonds in the thousands of proteins inside and outside cells:
control the dynamics of protein turnover
x Thermodynamics: Hydrolysis energetically favorable, Keq = 105. Therefore
proteolysis is irreversible (proteases are irreversible biological switches) and
must be stringently controlled. Default position of regulated proteases must be in
the off state:
x Diverse biological roles:
Signal transduction: Polypeptide hormone, growth factor activation
Proteolysis of INB to release NFNB to go from
cytoplasm to nucleus
Defense:
Blood coagulation and fibrinolysis (prothrombin and
fibrinogen vs TPA, plasminogen)
Cleavage of foreign proteins for MHC display
Development:
Fertilization; specification of dorsoventral polarity;
Retroviral protease cleavage of polyproteins
procollagen to collagen for self-assembly
Proliferation :
Proteolytic control of the cell cycle by cyclin
Degradation
Programmed Cell Death: homeostasis
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Catalytic Features
x Specificity: continuum from one bond cleaved in a protein substrate, e.g in
prohormone and procytokine activation, to digestive function on proteins.
Proinsulin o Insulin
ProIL1E o IL1E
x Rate Accelerations: up to 10 billion fold over nonenzymatic rates
x Mechanisms: four variants for activation of water molecules to attack and cleave
peptide bonds in protein substrates:
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Regulation and Control
x Temporal Control: produce proenzyme forms as inactive zymogen precursors,
cleavable and activatable on demand (e.g trypsinogen, procarboxypeptidase;
proacrosin; prococoonase)
x Spatial Control and Compartmentalization:
x 1. Protein inhibitors that bind tightly and produce inactive complexes on collision;
find these antiprotease proteins both outside cells (antitrypsin, antithrombin D2macroglobulin at 10-4M) and inside cells (IAPs)
2. Compartmentalization in organelles: lysosomal proteases for digestion of
endocytosed proteins
3. Compartmentalization in subcellular machines: proteasomes, cylinders with
protease active sites on the inside.
x Cascades of proteases: mechanisms for zymogen activation, amplification and
explosive bursts of proteolysis, both extracellular (blood coagulation) and
intracellular (caspases in programmed cell death)
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Producer Cells make Zymogen Forms of Proteases
Stomach Epithelia:
Pancreatic acinar cells:
Pepsinogen (aspartyl protease)
Chymotrypsinogen (serine protease)
Trypsinogen
(serine protease)
Proelastase
(serine protease)
Procarboxypeptidase (zinc protease)
Zymogen activation: controlled proteolytic clipping of inactive precursor to active form
of the protease: (single chain precursor to multi chain protease)
Trypsin-mediated activation of chymotrypsinogen in the intestinal tract
Autoproteolytic activation of HIV protease
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Protease Classification
(A) by site of action: exopeptidases (aminopeptidases, carboxypeptidases) vs
endopeptidases
(B) by reaction mechanisms and nature of active site residues involved in mechanism
x serine proteases
x cysteine proteases
x aspartyl proteases
x zinc (metallo) proteases
The serine and cysteine proteases act directly as nucleophiles to attack the substrate,
generatecovalent acyl enzyme intermediates. The aspartyl and zinc proteases activate
water molecules as the direct attacking species on the peptide bond.
Catalytic Mechanism of Chymotrypsin as a representative serine protease
Key Issue: How to accelerate peptide bond hydrolysis by 109.
Catalytic Triad: His57, Ser95, Asp102 typical inventory for serine proteases
His57: acts in sequential steps, as Base, then Acid Catalyst for proton transfers
Ser195: active site nucleophile, attacks peptide bond as ser alkoxide equivalent
Asp102: orients His57 side chain to H-bond to ser195
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Two Half Reactions:
1. Enzyme Acylation: Attack of ser195 on bound substrate’s peptide bond o
tetrahedral adduct o acyl enzyme and release of amine product
2. Enzyme Deacylation: Attack of OH- on acyl enzyme o tet adduct o acid
product
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General Acid and General Base Catalysis (Proton Transfers) to Lower Activation
Barriers and Accelerate Rates
Accessibility of His and His-H+ key for proton transfers in each step
Same Fundamental Mechanism for Cysteine Proteases where Cysteine thiolate
replaces serine alkoxide as active site nucleophile: Acyl enzyme intermediate is
Peptidyl-S-Enzyme
Specificity in Serine and Cysteine Proteases:
Given the conseved catalytic triad inventory in all the serine and cysteine proteases and
thus a common mechanism, how is specificity imposed?
x The Protease active sites have specificity pockets, subsites for binding the side
chain of the amino acid on the acyl side of the peptide bond to be cleaved. Many
proteases have even more extensive specificity subsites for recognizinf n+1,n+2
and/or n-1, n-2 residue side chains on protein substrates.
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x For example while the identity and placement of the Asp-His-Ser catalytic triad in
chymotrypsin, trypsin, and elastase is essentially identical, the specificity pockets
differ, accommodating a hydrophobic aryl side chain (Cht), a cationic side chain
(trypsin) or a small aliphatic side chain (elastase)
x The cysteine protease family (11 members) that act in apoptotic (programmed cell
death) cascades all recognize the anionic side chain of an Asp residue and so
cleave just downstream of such side chains, giving the name Caspase to this
family; Cysteinyl proteases that cleave at asp residues.
Aspartyl Proteases and Zinc Proteases Activate Water Molecules in the Active Site
for Direct Attack on the Protein Substrate’s Peptide Bond.
Aspartyl Protease Catalytic Logic:
x Two aspartate residues in the enzyme active site, disposed on opposite faces of
the peptide bond to be cleaved. One asp (Asp-COO-) acts as general base to
activate the attacking H-OH, the second asp (Asp-COOH) acts as general acid to
protonate the departing amine product. The two asps act in complementary
fashion for breakdown of the tetrahedral adduct.
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x The gastric protease pepsin has both asp residues in the same polype[tide chain
while the 99 residue HIV protease has a single Asp. It becomes active as a dimer,
providing the active site across the dimmer interface. This mechanism and
architecture guided the development of HIV protease inhibitors that mimic the
tetrahedral adduct.
Zinc Protease Catalytic Logic:
x One atiom of Zn++ coordinated specifically in active site to two his, one glu side
chain brings superacid metal divalent cation into active site, with two additional
coordination sites. In ground state, HOH bound and dissociates to Zn-OH, ready
to attack the substrate peptide bond.
x Once the protein substrate is bound the active site zinc can coordinate to the
carbonyl oxygen of the peptide bond to be attacked, lowering barriers
electronically for –OH attack. The tetrahedral adduct is coordinated to the active
site zinc. A conserved glutamate side chain in the active site now acts as catalytic
base to protonate the amine product as it leaves.
x Substrate analogs that have strong coordination sites for zinc (-SH, -COO) can be
ppotent, selective inhibitors of zinc proteases.
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Serine Proteases
x Size of the superfamily increases in eukaryotes: 1 gene in yeast, 18
genes in the worm, several hundred genes in Drosophila:
x Interest in proteomic approaches to identify function of the hundreds
of serine proteases. Activity-based profiling approaches using
covalent tethers attached to biotin for profiling (PNAS,96, 14694,
1999; Curr Opin Chem Biol 4, 663, 2000)
Cysteine Proteases
x Family of cathepsins (11 members) in lysosomes; cathepsin K in bone
remodeling, cathepsin L in antigen processing
x Family of 11 Caspases: Cysteine proteases that cleave at asp residues;
cascade of caspase action in programmed cell death (initiator,
amplifier, executioner proteases)
Aspartyl Proteases
x Pepsin the first example
x HIV protease, a dimer with one Asp/subunit. Target for anti AIDS
drugs: protease inhibitors such as saquinavir, ritonavir, indinavir
x E- and J-secretases in production of AE40 and AE42 in Alzheimers
plaques
Zn-Metalloproteases
x Angiotensin Converting Enzyme; ACE inhibitors for high blood
pressure control
x D-secretase in AE production
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Protease Case Study; DEJ- Secretases
D-secretase membrane-tethered metalloprotease
E-secretase membrane-anchored aspartyl protease
J-secretase oligomeric complex containing presenilins 1 and 2, aspartyl
proteases
Proteolysis of the Amyloid Precursor Protein
Tandem action of E- then J-secretase yields AE1-40 or 1-42 while action of
D-secretase blocks AE formation
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E-secretase catalyzes initial step in AE formation, likely pharmacologic
target. Xray structure allows structure-driven design of inhibitors;
mechanism allows ts analog design for E and J-secretases
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Biological role for members of the J-secretase family
The identification of J-secretase as an integral membrane protease puts this
protease in a family that also cleave SREBP in sterol metabolism and Notch
in developmental signal transduction pathways. Notch cleavage by Jsecretase releases the intracytoplasmic domain to go to the nucleus and
function as a transcription factor.
(Esler and Wolfe, Science 293, 1453, 2001)
More general biological roles for D-secretase protease family members
include ectodomain shedding of membrane proteins and receptors such as
TNF, the TNF p75 receptor, TGFD, and L-selectin adhesion molecules
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Caspase Cascades in Apoptotic Pathways:
x Metazoans eliminate cells that are damaged, mislocated, have become superfluous
x Activate an execution program of a cascade of controlled proteases, involving the
Caspase family.
x Various stimuli activate the program
Specific ligation of death receptors
Ionizing radiation’antineoplastic drugs
Withdrawl of growth factors
x Convergence to a common execution program
Signal at cell membrane
Signal transduction via receptors with Death Domains and Death Effcetor
Domains
Protease Cascade with initiator caspases, amplifier Caspases, executioner
Caspases that protelyze key intracellular protein substrates
Caspase Cascade represents intracellular signaling pathways via proteolysis, with
initiation, propagation, and termination phases.
Initiation phase: Caspases activated hierarchically by protein-protein interactions to
convert from single chain zymogens to two chain active proteases
Propagation phase: Proteolysis of proteins at aspartyl residues. Some of these are pro
forms of caspases, some are target proteins
Termination Phase: Difficult to terminate the cascade once full amplification is
underway. Low levels of active forms of Caspases can be blocked by intracellular
antiprotease (IAP) proteins that act as tight-binding pseudosubstrates for Caspases, with
picomolar to nanomolar Ki values.
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Virally infected cells are loci for running apoptotic programs and viruses are known to
produce caspoase inhibnitors to block the programmed cell death cascade: these include
CrmA protein and p35. The p35 protein is cleaved at an internal asp residue but the two
product fragments remain tightly bound and shut down the Caspase. The CrmA protein is
also a substrate with selectivity for Caspase 1 and 8 .
Cells at rest: caspase are shut off, sitting around as inactive zymogens
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