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Historical facts
• 1835 Berzelius J.J.: Theory of chemical catalyst
• 1860 Pasteur L.: yeast, catalyst boud to cells
• 1899 Buchner E.: Isolation of enzymes of alcoholic fermentation
• 1926 Summer B.J.: Preparation of plant urease in crystalline form
• 1930-1936 Northrop: Isolation of pepsin, trypsin and chymotrypsin in crystalline form
• The enzymes: proteins (99,9%)
Enzymes
• Biochemical, physiological role• Regulation of the enzyme activity• Therapy• Their clinical role
Catalyst
7Catalase
13Pt (colloidal)
18Without catalyst
Go kcal/moleH2O2 decompositon
DCBA
G & K
B*A
D*C(i)...K
nene
neneo
BA
DCRTlnΔGG (ii)..Δ
BA
DCRTlnΔG0 o
RTlogK*303,2ΔG
RTlnKΔGo
o
Exergon: spontaneously, G<0 K>1
Endergon: G >0 K<1
Enzymes: Common in mode of action
PEEPESSE
Enzyme: substrate binding side
Active site
Substrate
Enzyme activity: influence of temperature
t oC
Ac %
•Increase in temperature: increase of activity•Increase in temperature: heat denaturation
Enzymes: pH optimum
pH = 8 pH = 8pH = 2 pH = 6
Act
ivit
y
trypsin Cholinesterasepepsin papain
The lock and key; theory of the induced fit
+
Endopeptidases
Exopeptidases
Aminopeptidases Carboxipeptidases
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
The Chymotrypsin hydrolysis 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
Peptid
Hydrolysis of peptide bond
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
Chymotrypsin reacts with DIPF at Ser195 –OH
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 by 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 condition
Kinetics of chymotrypsin catalysis
(measured by synthetic substrate)
Hydrolysis of peptide bonds
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
Active site and its function of chymotrypsin
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 – short side chain residue i. e. .:
Ala, Ser
• Thrombin – Fibrinogen, V, VIII, PC• Plasmin - Fibrin,
Substrate binding site of chymotrypsin, trypsin & elastase
O OAsp 189 Val 216 Val 190
Chymotrypsine Trypsine Elastase
Cystein, aspartil and metalloproteases: peptide cleavage
NH NSH
X CO
R1
• Papaine – isolated form papaye
• Nucleofilic attack - peptide bond Catepsins – immunsystems
• Caspases - apoptose
Cistein, aspartil and metalloproteses: peptide bond cleavage
OO
O
O
HO
H
H
R1
CX
O
-
• Asp – protonated• Asp –
deprotonated
• H2O
• Renin• Pepsin
Cistein, aspartil and metalloprotease: peptide cleavage
B O
H
H Zn2+
X CO
R1
• Bacterial termolysine
• Carboxypeptidase A• Zinc proteases
(matrix metalloproteinases MMP)
• MMP – tissue degradation and remodelling
Catalytical mechanism of acetylcholinesterase
Enzyme Ser OH
CH3 C
O
O CH2 CH2 N+
CH3
CH3
CH3
OH CH2 CH2 N+
CH3
CH3
CH3Enzyme Ser O C CH3
O
OH2
Enzyme 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 – Tokyo-i tube• Tabun • Chemical weapon• légzésbénulás• Parathion – rovarirtó• Rádioaktív DIFP egér izom
diagfragma végkészülék: 12000 / mm2 receptor
PO
F
CH
CH3
CH3 CH3
PS
O
OCH2
O NO2
CH3
CH2CH3
Physostigmine (Eserin)
Physostigmine carbamoylates the Ser-OH of the acetylcholinesterase
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
The rate of hydrolysis of the carbamoyl
enzyme intermedier is low
Trial by ordeal
Neostigmine
Neostigmine carbamoylates the Ser-OH of acetylcholine esterase
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
Inhibition of acetylcholine esterase•[acetylcholin] enhancement;•Intraocular pressure - decreased
A gyomor és hasnyálmirigy zimogénjei
Szerv Zimogén Aktív enzim
Gyomor Pepszinogén Pepszin
Hasnyálmirigy Kimotripszinogén Kimotripszin
Hasnyálmirigy Tripszinogén Tripszin
Hasnyálmirigy Prokarboxipeptidáz Karboxipeptidáz
Hasnyálmirigy Proelasztáz Elasztáz
Enteropeptidáz
Tripszinogén Tripszin
Proelasztáz ProkarboxipeptidázElasztáz Karboxipeptidáz
Kimotripszinogén ProlipázKimotripszin Lipáz