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