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