Post on 19-Jan-2016
description
Key and lock; theory of induced fit
+
Endopeptidase
ExopeptidaseAminopeptidase Carboxypeptidase
Proteolytic activation of zymogensProteolytic activation of zymogens
Intestine Pancreas
Trypsinogen
Chymotrypsinogen
- Chymotrypsin
Trypsin
Enteropeptidase
- Chymotrypsin
Trypsin inhibitorInactive trypsine
1……………………………………………………………………..245
Chymotrypsinogen
16…………………………………………..2451…………..15
Trypsin-Chymotrypsin
Chymotrypsin
16…………………….146 149………2451……….13
-Chymotrypsin
Chymotrypsin: hydrolysis of peptide and ester bonds
R1
O
OR2 OH2 R1 C
O
OOH R2+ +
Protease
Ester
R1
O
NH
R2 OH2 R1 C
O
ONH3
+R2+ +
Protease
Peptide
Hydrolysis of peptide bonds
Hydrolysis of ester bonds
Substrate specificity of Chymotrypsin
CH3
NH3
+
H
NH
NH
O H
OCH2
O
NH2
C
NH
C NH
CH
CH2
OH
H
C
CH2
CH2
S
CH3
C
NH
C
C
O
O
H
OH
OOCH2
CH2
C NH2
O
Ala Phe Asn Ser Met Gln
• Phe•Typ•Trp•Met
Ser195 –OH of chymotrypsin reacts with DIPF
CH3
CH3
CH
O
P
O
CH3
CH3
H
F
O
CH3
CH3
CH
O
P
O
CH3
CH3
H
O
OCH2OH
Ser 195 Ser 195
CH2
FH
Kinetics of chymotrypsin catalysis (measured with synthetic substrate)
CH3
C
O
NH
C
CH2 H
C
O
O
NO
O
OH2
CH3
C
O
NH
C
CH2 H
C
O
OHO
NO
O
H+
+ +
Time [milisecundum]
A
Burst phase
Steady state
Kinetics of chymotrypsin catalysis (measured with synthetic substrate)
Two stages of the enzyme action
OH X C
O
R1
XH
+
AcylationO C
O
R1OH2
Deacylation
OH C
O
R1
OH+
Enzyme Acyl-enzyme Enzyme
E + S ES EP2 E
P1P2
Chymotrypsin active site & its function
N NHO
O
OH
Asp 102 His 57 Ser 195
N NHO
O
OH
Asp 102 His 57
Ser 195
+
Alkoxid ion
OHN NHO
O
R2NH
C
O
R1
R2NH
C
O
R1
OHN NHO
O+
N NHO
O
H NR2
H
C
O
R1
O
H
R2N
HOHN NHO
O
CR1
R2NH
O
Hydrolysis of peptide bond – by chymotrypsin
O
C
O
R1OH
HN NHO
O
+
H
R2N
H
N NHO
O
C
O
R1O
OH2
N NHO
O
C
O
R1O
OH
H
Acyl – enzyme intermedier
Acyl – enzyme intermedier
Tetrahedral - intermedier
Hydrolysis of peptide bond – by chymotrypsin
Hydrolysis of peptide bond – by chymotrypsin
C
O
R1OH
N NHO
O
HO
+
N NHO
O
OH
R1C
OH
O
OHN NHO
O
R2NH
CR1
O
Homolog enzymes
• Chymotrypsin – Phe, Tyr, Trp, Met
• Trypsin - Arg, Lys
• Elastase – amino acids with short side chain residues ie.: Ala, Ser
• Thrombin – Fibrinogen, V, VIII, PC
• Plasmin - Fibrin,
Substrate bingind site S1 of chymotrypsin, trypsin & elastase
O OAsp 189 Val 216 Val 190
Chymotrypsin Trypsin Elastase
Cystein, aspartyl & metalloproteases
NH NSH
X CO
R1
• Papain isolated from papaya
• Nucleophyl attack on peptide bond
• Cathepsins – immunsystem
• Caspases - apoptoses
Cistein, aspartyl & metalloproteases
OO
O
O
HO
H
H
R1
CX
O
-
• Asp – protonated• Asp – deprotonated
• H2O
• Renin• Pepsin
Cistein, aspartyl & metalloproteases
B O
H
H Zn2+
X CO
R1
• Bacterial termolysine
• Carboxypeptidase A
• Zinc proteases (matrix metalloproteinases MMP)
• MMP – tissue degradation and remodelling
Cholinerg synapsis
Presynaptical membrane
Postsynaptical membrane
OH CH2CH2N+
CH3
CH3
CH3
CH3 C
O
S CoA
SH CoA
CH3 C
O
O CH2 CH2 N+
CH3
CH3
CH3
Choline acyltranspherase
Acetylcholine esterase
• Synaptic cleft
• Bound to collagen GAG network
• Hydrolysis of Acethylcholine
22
• Turnovernumber: 25000 / sec
Acetilkolin receptor
Catalytic mechanism of acetylcholine esterase
Enzim Ser OH
CH3 C
O
O CH2 CH2 N+
CH3
CH3
CH3
OH CH2 CH2 N+
CH3
CH3
CH3Enzim Ser O C CH3
O
OH2
Enzim Ser OH
CH3 C
O
O
CH3
CH3
CH
O
P
O
CH3
CH3
H
F
O
Organic phosphate inhibitors of acetylcholine esterase
PO
CN
N
CH3
CH3
OCH2CH3
• Sarin – Metro of Tokyo
• Tabun
• extremely toxic substance • it is classified as a
weapon of mass destruction
• Parathion – pesticide
• Labelled DIFP
• 12000 / m2 receptor/mouse diafragma
PO
F
CH
CH3
CH3 CH3
PS
O
OCH2
O NO2
CH3
CH2CH3
Physostigmin (Eserin)
Carbamoylation of Ser-OH in acetylcholine esterase
NN
CH3
CH3 CH3
OCNH
CH3
O
NN
CH3
CH3 CH3
OCNH
CH3
OEnzim Ser OH
Enzim Ser O C NH
CH3
O
OH2
Enzim Ser OH
NN
CH3
CH3 CH3
OH
CH3 NH
C O
O
Slow hydrolysis of carbamoyl-enzyme
intermedier Ordeal
In witchcraft trials
Neostigmin
Carbamoylation of Ser-OH in acetylcholine esterase by neostigmin
N+
OCN
CH3O
CH3
CH3
CH3
CH3
OCN
CH3O
CH3
N+
CH3
CH3
CH3
Enzim Ser OH
Enzim Ser O C NCH3
O
CH3
OH2
Enzim Ser OH
OH N+
CH3
CH3
CH3
CH3
N C O
O
CH3
Slow hydrolysis of carbamoyl-enzyme
intermedier: it inhibits acetylcholine esterase, [acetylcholine] increase;Decrease in intraocular
pressure
(CH3)3 N+
(CH2)10 N+
(CH3)3
(CH3) N+
CH2CH2O C
O
CH2CH2C
O
O CH2CH2N+
(CH3)
Dekametonium: bounds to receptorConstant depolarisation
Succinylcholine: slow hydrolysisPseudocholine esterase
Zymogens of pancreas
Organ Zymogen Active enzyme
Stomach Pepsinogen Pepsin
Pancreas Chymotrypsinogen Chymotripsin
Pancreas Trypsinogen Trypsin
Pancreas Procarboxypeptidase Carboxypeptidase
Pancreas Proelastase Elastase
Enteropeptidase
Trypsinogen Trypsin
Proelastase ProcarboxypeptidaseElastase Carboxypeptidase
Cymotrypsinogen ProlipaseCymotrypsin Lipase