Transcript Proteases - Home - KSU Faculty Member websites

King Saud University
College of Science
Department of Biochemistry
Disclaimer
• The texts, tables and images contained in this course presentation
are not my own, they can be found on:
– References supplied
– Atlases or
– The web
Mechanism of Enzyme Action
BCH 321
Professor A. S. Alhomida
King Saud University
College of Science
Department of Biochemistry
Disclaimer
• The texts, tables and images contained in this course presentation
(BCH 320) are not my own, they can be found on:
– References supplied
– Atlases or
– The web
Mechanism of Enzyme Action
Proteases
Professor A. S. Alhomida
2
Proteases (or peptidases)
Enzymes that catalyze the breaking of the
amide peptide bond (proteolysis) by hydrolysis.
Scissile bond
P1'
O
O
N
H
P1
+
H2O
P1'
O
+
H3N
+
P1
3
Proteases are essential to
physiologic processes
•
•
•
•
•
•
•
•
•
Inflammation
Infection
Fertilization
Allergic reactions
Cell growth and death
Blood clotting
Tumor growth
Bone remodeling
Etc…
4
Proteolytic enzymes are divided into
two different categories
• Limited proteolysis: a protease cleaves only one or a
limited number of peptide bonds of a target protein
leading to the activation or maturation of the formerly
inactive protein.
– e.g. conversion of prohormones to hormones.
• Unlimited proteolysis: in which proteins are degraded
into their amino acid constituents.
– Ubiquitin/proteasome pathway: First conjugated to multiple molecule of
the polypeptide ubiquitin. This modification marks them for rapid
hydrolysis by the proteasome in the presence of ATP.
– Lysosome pathway: Proteins are transferred into lysosomes which
contain proteases that completely digests the protein into the amino
acids.
• These unlimited proteolysis pathways are essential for protein
quality control and the reuse of amino acids during starvation.
5
Some Cells Secrete proteases
• Proteases are secreted by cells into the
surrounding tissues to cause the
destruction of proteins in extracellular
material.
• Secreted into an area (stomach) for the
breakdown of protein in the diet.
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Protease, peptidase, or proteinase?
• Protease: is synonymous with peptidase.
• Subclass EC 3.4.). 3 = hydrolase 4 = amide bond (peptide bond)
• The Peptidases are comprised of two groups of enzymes:
– Endopeptidases: cleave peptide bonds at points within the protein.
– Exopeptidases: remove amino acids sequentially from either N or Cterminus.
– Proteinase: synonymous with endopeptidase.
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The EC nomenclature for proteases
•
•
•
•
•
•
•
•
•
•
•
•
3.4.11 Aminopeptidases3.4.13 Dipeptidases
3.4.14 Dipeptidyl-peptidases and tripeptidyl peptidases
3.4.15 Peptidyl-dipeptidases
3.4.16 Serine-type carboxypeptidases
3.4.17 Metallocarboxypeptidases
3.4.18 Cysteine-type carboxypeptidases
3.4.19 Omegapeptidases
3.4.21 Serine proteinases
3.4.22 Cysteine proteinases
3.4.23 Aspartic proteinases
3.4.24 Metallo proteinases
3.4.99 Proteinases of unknown mechanism
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The four mechanistic classes of proteases
• Serine proteases
• Cysteine proteases
• Aspartic proteases
• Metallo proteases
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Serine protease catalytic mechanism
Serine Proteases often use the
chymotrypsinogen numbering:
His 57, Asp 102 and Ser 195
Two distinct evolutionarily unrelated families:
(The classic example of convergent evolution)
• Mammalian: chymotrypsin, trypsin or
elastase
• Bacterial :subtilisin.
Ser/His/Asp Catalytic triad mechanism
Ser: nucleophile
His: general base
Asp: help orient and neutralizes charge on His
Covalent tetrahedral intermediates
Covalent catalysis
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Famous serine proteases
•
•
•
•
•
•
•
•
•
Trypsin
This serine endopeptidase is the active form (MW 24,000) of the pancreatic proenzyme trypsinogen that
is activated in the intestine by enterokinase.
Chymotrypsin
A group of serine proteases found in pancreatic secretions that hydrolyze peptide bonds in the process of
digestion. This endopeptidase is made in the exocrine pancreas as a precursor that is activated by
proteolytic cleavage in the duodenum by trypsin.
Elastase
MW 25.000. Serine protease of broad specificity made in the exocrine pancreas.
Subtilisin
Serine protease of bacterial origin.
Plasmin
Serine protease derived from the precursor plasminogen that breaks down insoluble fibrin thus disolving
blood clots.
Thrombin
Serine protease present in plasma as a precursor called prothrombin (MW 72,000). The active form (MW
34,000) converts plasma fibrinogen into insoluble fibrin forming the basis of a blood clot.
Kallikrein
Causes the liberation of kinins from plasma protein precursors causing vasodilation and possibly
hypotension.
Streptokinase
Protease produced by hemolytic bacteria that activates plasminogen thus producing plasmin which
disolves fibrin. Therapeutically streptokinase has been used to dissolve pulmonary emboli and venous
thromboses.
Urokinase
This protease is found in human blood and urine. It activates plasminogen by the cleavage of a
propeptide forming the active serine protease plasmin. Therapeutically it is used to dissolve blood clots.
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The Serine Proteases
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•
•
•
•
Trypsin, chymotrypsin, elastase, thrombin,
subtilisin, plasmin, TPA
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 locations
of the three crucial residues differ somewhat
Enzymologists agree, however, to number them
always as His-57, Asp-102, Ser-195
Burst kinetics yield a hint of how they work!
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Serine Protease Mechanism
•
•
•
•
•
A mixture of covalent and general acid-base
catalysis
Asp-102 functions only to orient His-57
His-57 acts as a general acid and base
Ser-195 forms a covalent bond with peptide
to be cleaved
Covalent bond formation turns a trigonal C
into a tetrahedral C
The tetrahedral oxyanion intermediate is
stabilized by N-Hs of Gly-193 and Ser-195
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Artificial Substrate for Serine
Proteases
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Assay for Serine Proteases
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Identification of Catalytic
Residue Ser-195
19
20
21
Identification of Catalytic
Residue His-57
(N-tosyl-L-phenylalanine
chloromethylketone)
22
1. Conformational distortion forms the tetrahedral
intermediate and causes the carboxyl to move close to the
oxyanion hole
2. Now it forms two hydrogen bonds with the enzyme that
cannot form when the carbonyl is in its normal conformation.
3. Distortion caused by the enzyme binding allows the
hydrogen bonds to be maximal.
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25
Serine proteases
• Several different families - all have Ser in
active site and all have the same reaction
mechanism.
• The two most commonly-studied are:
•
Trypsin family
•
Subtilisin family
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Trypsin family of Serine Proteases
• Includes:
•
trypsin
•
chymotrypsin (used for numbering)
•
elastase
•
thrombin
•
coagulation enzymes
•
plasmin
•
complement C1r and C1s
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Serine proteases
• Synthesized in zymogen (inactive) form
and activated by cleavage of specific
peptide bonds.
• Catalytic triad:
•
•
Serine 195
Histidine 57
Aspartate 102
• Numbering based on chymotrypsin.
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Zymogen activation (Lehninger 8-31)
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Chymotrypsin structure (Lehninger Fig 8-18)
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Chymotrypsin
Diagram from
Branden &
Tooze, 1991
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Key residues
• Ser 195 (1/28) labelled with DFP
(diisopropylfluorophosphate)
• His 57 labelled with Tosyl-Phechloromethyl ketone
• Labelling of these residues inactivates
serine proteases.
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Protease - key areas
• Binding
• Main chain binding (non-specific)
• Specificity pocket (residue 189)
• Catalysis
• active site - charge relay system
• oxyanion hole formed
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Specificity pocket explains protease
specificity (residues 189, 216 and 226)
Chymotrypsin
aromatic
Trypsin
basic
Diagram from Branden & Tooze, 1991
Elastase
small, uncharged
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Substrate modification (Lehninger)
Small substrates for chymotrypsin
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Two domains and other key residues
Active site, S195,
H57 and D102, lies
between domains in
chymotrypsin
oxyanion
hole (193195)
main chain
substrate
binding
(214-216)
substrate
specificity
pocket
(189,216,226)
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Branden & Tooze, 1991
Oxyanion Hole Role
•
•
•
•
Stabilizes the tetrahedral transition state
Form H-bonds
Uses catalytic triad plus Gly 193
Positions and orient substrate for
hydrolysis
• Stabilizes the TS
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Zymogen to active
A: zymogen
Chymotrypsinogen
B: active
chymotrypsin
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Subtilisin family (SB clan)
• Includes furin, PC2 and PC3 - all active in
prohormone processing
• No sequence identity with trypsin family
• Structure quite different (a/b structure;
trypsin is b-barrel)
• But - same reaction mechanism and
organisation of catalytic triad
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Serine protease families convergent evolution
Trypsin family (H57, D102, S195)
His
Asp
Ser
Subtilisin family (D32, H64, S221)
Asp His
Ser
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Chymotrypsin and subtilisin
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Different structures but similar active sites
Mechanism of Serine Protease
His -57
H
102
Asp
N
O
O
N
O
195
Ser
N
N
H O
O
H
C
N
C
N
O
O
H
R2
H
O H
R2
R1
S1 R1
H
O
O
N
N
102
H
O H
H
N
C O
R1
Tetrahedral Intermediate -1
O
H
H2O
Oxyanion Hole
P1
O
H
C O
R1 Acyl-Enzyme
H
H
N H
O
O
H O
H
R2 N
N
O H
N
O
O
R2
N
O
C
O
O
N
N
H
O
O
R1
Tetrahedral Intermediate-2
C OH
R1
P2
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Cysteine protease catalytic mechanism
Papain is the archetype and
the best studied member of the
family
1
4
Cysteine proteases sometimes
use the Papain numbering
system: Cys25 and His159.
Asn175 helps stabilize the
Cys- and His+ ion pair
Unlike Ser protease Cys
already ionized before
substrate binding.
a priori activated enzymes.
2
3
Cys25 and His159 play the same
catalytic roles as Ser195 and His57
respectively in the serine proteases.
Covalent tetrahedral intermediates
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Famous cysteine proteases
• Papain
Thiol protease from the papaya. MW of about 23,400 used for
tenderizing meats.
• Bromelain
Thiol protease found in pinapple juice and stem tissue. Glycoprotein
of about 33,000 MW used in meat tenderizing, beer production and
hydrolization of proteins in the food industry.
• mammalian lysosomal Cathepsins (11)
These intracellular cysteine proteases are involved in such diverse
activities as blood clotting, cancer growth and metastasis and bone
remodeling. Several varieties have been isolated from various
tissues, each having specific functions.
• cytosolic calpains (calcium-activated)
• parasitic proteases (e.g Trypanosoma).
• Interleukin-1-beta Converting Enzyme
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Aspartic protease catalytic mechanism
Bi-lobe enzymes with the active site
located between two homologous lobes.
1
Asp general acid-base catalysis
2 "push-pull" mechanisms (steps 1 and 3)
Non-covalently bound (neutral) tetrahedral
intermediate.
Low-barrier H-bonds may be involved.
3
45
Famous aspartic proteases
• Pepsin
Aspartic protease that cleaves bonds involving phenylalanine and leucine
preferentially. MW 34,500. This endopeptidase is the principal digestive
enzyme in gastric juice formed from the precursor pepsinogen (MW
38,000). (first protein crystals to diffract)
• Renin
Also known as angiotensinogenase. It is made by the juxtaglomerular
cells in the kidney, released into the blood stream and acts to convert
angiotensionogen into angiotensin I. It is an aspartyl protease having a
molecular weight of about 40.000 Renin is elevated in some forms of
hypertension.
• cathepsins D
• fungal proteases (penicillopepsin, rhizopuspepsin, endothiapepsin).
• viral proteinases such as HIV protease (retropepsin).
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The Aspartic Proteases
•
•
•
•
Pepsin, chymosin, cathepsin D, renin and
HIV-1 protease
All involve two Asp residues at the active site
Two Asps 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
symmetry
HIV-1 protease is a homodimer
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Aspartic Protease Mechanism
The pKa values of the Asp residues are crucial
• One Asp has a relatively low pKa, other has
a relatively high pKa
• Deprotonated Asp acts as general base,
accepting a proton from HOH, forming OHin the transition state
• Other Asp (general acid) donates a proton,
facilitating formation of tetrahedral
intermediate
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Asp Protease Mechanism
• What evidence exists to support the
hypothesis of different pKa values for the
two Asp residues?
• Bell-shaped curve is a summation of the
curves for the two Asp titrations
• In pepsin, one Asp has pKa of 1.4, the
other 4.3
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Mechanism of Aspartic
protease
O
O
25`-Asp
O
H
O
O
H
C Proline
N
Aromatic
Asp-25
O
H
H
S1
O
O
25`-Asp
O
H
N
Asp-25
HO
O
H
O
C R1
H
R2
Tetrahedral Intermediate
R2
H
O
O
25`-Asp
OH
N
O
H
+
OH
C
R1
Asp-25
O
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HIV-1 Protease
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•
•
•
•
A novel aspartic protease
HIV-1 protease cleaves the polyprotein
products of the HIV genome
This is a remarkable imitation of mammalian
aspartic proteases
HIV-1 protease is a homodimer - more
genetically economical for the virus
Active site is two-fold symmetric
Two Asp residues - one high pKa, one low pKa
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Therapy for HIV?
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•
•
•
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
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Metallo-protease catalytic mechanism
• Most contain a Zn atom that is involved in catalysis.
Sometimes Zn can be replaced by Co or Ni.
• Zn is usually coordinated by 2 His and 1 Glu.
Many contain the sequence HEXXH
H
• Non-covalent (oxyanion) tetrahedral intermediate.
After the attack of a Zn-bound water molecule
on the carbonyl group of the scissile bond.
The intermediate breaks down by transfer of a proton
From a Glu to the leaving group (not shown).
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Famous metallo-proteases
• Thermolysin
Extracellular thermostable zinc metalloprotease
from bacterial sources.
• Collagenase
Proteases that breakdown collagen that are
produced by some species of bacteria such as
Clostridium. Also isolated in human wound
tissue, skin, bone, leukocytes, and cornea.
Family of metalloproteases (MW 68,000125,000) that require zinc and calcium for
activity. They are used in research for the
isolation of cells from animal tissues.
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Mechanism of Thermolysin
His Glu
143
Glu
His
BH
Zn2+
O
C
O
H
O
H
N
C
N
C
O
H
O
Zn2+
His-231
R2
OH
HO
R1
OH
HO
O
N H2
Zn2+
O
H
N
R2
R1
H
R1
Tetrahedral Intermediate
C
N
C
H
R2
N
Zn2+
C
N
N
C
N
H
O
O
H 2O
HO
OH
O
C
O
H
H
N
N
H
R2
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Mechanism of Thermolysin
• Zn2+ binds the amide carbonyl group
• Zn2+ polarizes the carbon of carbonyl
group
• The nucleophile water (OH-) is activated
by Glu-143
• His-231 is the acid for protonating the
leaving group (R1-NH2)
63
The Schechter and Berger
nomenclature for the description
of protease subsites
64
Scissile bond and Subsite nomenclature
solvent
solvent
P1’
P2
scissile bond
P3
P1
I144
F84
D142
I86
V132
I101
I86
P87
S88
S90
K145
Y143
I144
L95
M91
S1
S3
S1
peptidase
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Nucleophilic attack from Si vs. Re face
P 1'
N
7H
P1'
8
O
P 1'
O
6
P1
si-face
Ser/Lys protease P1
O
N
H
N
H
P1
re-face
Ser/His/Asp proteases
66
Angle of nucleophilic attack on a carbonyl
The Dunitz-Bürgi angle (105°)
From analysis of small molecule high resolution crystal structures
it was observed that: The preferred O…C=O angle is 105°
P1'
O
105
From inhibitor and substrate co-crystal structures:
The nucleophiles in the active-site of proteases
(Ser hydroxyl Og, Cys Thiol Sg, waters)
Are often orientated with respect to the scissile carbonyl
close to the Dunitz-Bürgi angle (105°)
N
H
O
P1
Ser
67
Evolutionary Classification of proteases
• MEROPS: Protease Data Base
– Families: Proteases with statistically
significant similarities in amino acid sequence.
– Clans: Protease families that are thought to
have common evolutionary origins.
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Proteases as research tools
• Limited proteolysis / sequencing / MS
– Protein crystallization
– Protein mobility (dynamics)
– Protein-Protein interactions
• NickPred: Web tool to understand limited proteolysis using PDBs
•
PROWL: A collection of protocols for limited proteolysis combined with Mass
Spec
69
Mechanism of Electron Transfer
in Biological System
1. Two One Electron Steps Mechanism
2. One Two Electron Steps Mechanism
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Two One Electron Steps
Mechanism
• A free-radical intermediate must form
which highly reactive and very energetic
species
• Discrete long-lived radical intermediates
are infrequently seen in biological systems
• Examples: flavin-Coenzyme
• Vit C, E, K, CoQ
• Metalloenzymes
71
One Two Electron Steps
Mechanism
1. Hydride (H-:) Mechanism
2. H+ Abstraction Mechanism
Most of enzyme hydrogenases are
enzymatic dehydrogenation where H- ion
transfer
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One Two Electron Steps
Mechanism
Hydride
Mechanism
X
Y
H
C
H
+ X
C
HY + X
C
Carbonium Ion
Proton
Abstraction
Mechanism
X
C
H
Y
H
+ X
C
HY + X
C
Carbonanion Ion
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