1

• PROTEAZE• TRIOZA FOSFAT-IZOMERAZA• LAKTAT DEHIDROGENAZA• DNA-METIL TRANSFERAZA

PROTEAZE

tripsinogen

tripsin

aktivacijakimotripsinogena

aktivacijaproelastaze

aktivacijaprokarboksipeptidaze

enteropeptidaza autokataliza

SERINSKE PROTEAZE KIMOTRIPSIN

2

Ser195

His57

Asp102

1. Zakaj je Ser195 tako reaktiven?2. Kako se S veže na E?3. Različna specifičnost.4. Aktivacija zimogena.

3

H2C

C

O

O

Asp102

N N

H2C

His57

CH2

OHH

Ser195

H2C

C

OH

O

Asp102

N NH

H2C

His57

CH2

O

Ser195

0.28 nm 0.30nm

CHO N

H

CH

CH2

C

O

O H

NH X

NH

Ser 214Gly 193

Ser195

N-formil triptofan

Gly216

Ser189

Gly226H--H

OHl

CH2l

kimotripsin Gly226

Asp189

Gly216H--H

COO-

lCH2

l

tripsin

Thr226Val216

Ser189

CH3

CH-

OH

CH3

-CH

CH3

OHl

CH2l

elastaza

4

Ile16

Ile16Asp194

Asp194

His157His157

Asp102Asp102

Ser195Ser195

superimpozicija kimotripsinogen/kimotripsin

Asp102Asp102

His157His157

Ser195Ser195

Ser189

Ser189

Gly226Gly226

Gly216 Gly216

5

P

O

O F

O

CH

CH

H3C

H3C

H3C CH3

DFPdisopropilfluorofosfat

C C

N

N

H

H

H

H

H

S

O

O

F

APMSF(4-amidino-fenil)-metan-sulfonil fluorid

6

TPCKtozil fenilalanil klormetil keton

CH3

S OO

NH

CH C CH2H2C Cl

O

inaktivira kimotripsinne inaktivira tripsin

CH3

S OO

NH

CH C CH2(CH2)4 Cl

O

H2N

TLCKtozil lizil klormetil keton

inaktivira tripsinne inaktivira kimotripsin

CH3

S OO

NH

CH CH2C

O

O C2H5

substrat

mestohidrolize

*AcNH Lys14

CO2H

NHAc

*Ac2O

H2N Lys14

CO2H

NH2Ac2O

AcNH Lys14

CO2H

NHAc

AcNH CO2H + H2N Lys14

CO2H

NHAc

acetiliran kimotripsinogen

acetiliran δ kimotripsinogen

aktivacijatripsin

Asp102His57

Ser195

Asp102

Asp102 Asp102 Asp102

Asp102

Asp102Asp102

His57

His57 His57 His57

His57

His57His57

Ser195

Ser195 Ser195 Ser195

Ser195

Ser195Ser195

7

C

O

NH

N..

Y-H

X+

nukleofilna skupina

pozitivni naboj

donor protona

METALOPROTEAZE KARBOKSIPEPTIDAZA A

Zn2+

His69

His196

Glu72

Tyr248Arg145 Glu270

8

NH2 CH

Rn+1

NH2 CH

Rn+1Rn CH

C

O

OH

+ CHRn

C

O

OH

+

Rn CH

C

O

NHCH Rn+1

CO

O-O

H

H

Zn2+

Glu270O-

HO

Rn CH

C NHCH Rn+1

CO

O-

Zn2+

Glu270

Zn2+

CO

O-

Glu270

H

CO

O-O

H

Zn2+

Glu270

CISTEINSKE PROTEAZE KATEPSIN K

His162Cys25

acilacija deacilacija

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ASPARTATNE PROTEAZE KATEPSIN D

Asp33 Asp231

substrati• fluorogeni substrati

– peptidil naftilamidni– metil kumarinski– EDANS-DABCIYL

• edans=(5-[(2’aminoetil)-amino] naftalensulfonska kislina• dabcyl =4-[[(4’-(dimetilamino)] fenil]azo] benzojeva kislina

• kromogeni substrati– peptidil naftilamidni– para-nitroanilidni C O

NH

CH CNH(CH2)3C

H2N

HNO

NH NO2

Bz-Arg-pNA (L-BAPA)

p-nitroanilid

benzoilna sk.

arginil

H2N

C

HN

NH(CH2)3HC

NH

C

C OCH2O

O

NH O O

H3C

Z-Arg-MCA

7-amino-4-metilkumarin

benziloksikarbonilna sk.

10

inhibitorji

• aminopeptidaze (amastatin, bestatin)• cisteinske in serinske proteaze (antipain)• serinske proteaze (APMSF, benzamidin,

kimostatin, DFP, DCI, TPCK, TLCK)• aspartatne proteaze (pepstatin)

H2N CH CH

OH

CH2

CH

CH3H3C

C

O

NH CH

CH2

C NH

CH

CH3H3C

O

CH

CH2

C

CH

CH3H3C

O

NH CH C

OH

O

CH2

C

OH

O

CH2 CH

OH

C

O

NH CH C

CH2

OH

CH

H3C CH3

O

bestatin

amastatin

HOOC CH NH

CH2

C

O

NH CH

(CH2)3

C

NH

CHN NH2

O

NH CH

CH2

C

O

NH CH

CH

H3C CH3

C

H

O(CH2)3

NH

C

NH2H2N

antipain

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P

O

O F

O

CH

CH

H3C

H3C

H3C CH3

HOOC CH NH C NH CH C NH CH C

OO

NH CH C

H

OO

CH2 CH2 CH2

NH

HN

CH

NH2 CH3H3C

C C

N

N

H

H

H

H

H

S

O

O

F

C

NH2HN APMSFamidino-fenil-metana-sulfonil fluorid

benzamidin

kimostatin

DFPdisopropilfluorofosfat

O

O

Cl

Cl

DCIdikloroizokumarin

C NH

O

CH2

CH

CH3H3C

CH C

CH

CH3H3C

O

NH CH

CH

H3C

C

CH3

O

NH CH

CH2

CH

CH3H3C

CH

OH

CH2

C O

NHHC

CH3

C

O

NHCHCH

CH2

CH

H2C

OH

H3C CH3

HOOC

pepstatin

TRIOZA FOSAT-IZOMERAZA

12

CHO

C

CH2OPO32-

H OH

G3PD-gliceraldehid 3-fosfat

CH2OH

C

CH2OPO32-

O

DHAPdihidroksi aceton fosfat

trioza fosfat-izomeraza

[ ][ ]DHAP

K= =367G3P

Asn14

Asn14

Asn78

Asn78

Glu165

Glu165

His95

His95

Lys12

Lys12

His95

Lys12Glu165

13

zanka 166-176

zanka 166-176Glu165

Glu165

Lys12Lys12

His95His95

PGA

C

C

CH2OPO32-

OHH

H O

C

C

CH2OPO32-

OH3H

H

O

C

C

OH

CH2OPO32- OH

C

C

OH

CH2OPO32- OH

B H+

B 3H+

izmenjavaz 3H2O

cis-enediol

B:

B:

CO

O

C

C

O

H2C

OP

O OO

N

N

H

H OH

H

G3P

Glu165

Lys12

His195

CO

O C

O H

OH

H2C

OP

O OO

N

NH

H

cis enediolintermediat

Glu165

Lys12

His195

CO

O

C

C

H

H

O H

O

H2C

OP

O OO

N

N

H1

2

3

DHAP

Lys12

Glu165

His195

CO

O

C

C

O

OH

H2C

OP

O OO

N

NH

H

H

Glu165

Lys12

His195

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LAKTAT-DEHIDROGENAZA

CH3

C

COOH

O

CH3

C

COOH

H OH

laktat piruvat

laktat dehidrogenaza+ NAD+ + NADH

+ ++laktat +piruvat+ NAD NAD NADH NADHlaktat piruvat-laktat -piruvat

E+NAD E E E E E+NADH⎯⎯→ ⎯⎯⎯→ ⎯⎯→ ⎯⎯⎯→ ⎯⎯→←⎯⎯ ←⎯⎯⎯ ←⎯⎯ ←⎯⎯⎯ ←⎯⎯

5 10 15 ms

NA

DH

/ m

ol a

ktvn

ega

mes

ta

15

N

N N

N

NH2

O

OH OH

CH2 O P O P O CH2

ON

C

O

NH2

OH OH

O O

O OAsp53

Tyr85

Asp30Lys58 Ala98 Glu140

Lys250

Val27, Val52Val54, Ile96Ala98, Ile119Ile123

Gly28Met55

Val32Val247

Val32Thr97Pro139

Glu140Lys58 Arg101

NAD+

Arg171

Arg109His195

SzankaAla98-Phe120

NC

O

NH2

C

C

OH

H3C

O O

H:N N

CH2

H

CH

NH

NH2H2N

(CH2)3

C NH (CH2)3

H2N

H2N

His195

Arg109

Arg171

NAD

laktat

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DNA METIL-TRANSFERAZA

proteini, ki se vežejo na DNA1. vijačnica-zavoj-vijačnica proteini

(helix-turn-helix proteins)

2. Proteini s koordinativno vezanim cinkom (zink-coordinating proteins)

3. proteini z levcinsko zadrgo (zipper-type proteins)

4. preostali α-vijačni proteini (other α-helix proteins)

5. proteini z β-ploskvijo (β-sheetproteins)

6. proteini z motivom β-lasnice in trakom (β-hairpin/ribbon proteins)

7. drugo8. encimi

HhaI DNA-metiltransferaza

velika domena 1-93, 304-327

vmesna povezava 276-303

mala domena 194-275

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domena za prepoznavanje tarčehipervariabilna regija231-253(target recognition domain)

Ado Met(S-adenozil-L-metionin)

mehanizem reakcije

karbanionski intermediat

N

NO

H

NH2

DNA

6

5N

NO

H

NH2

DNA

6

5

S-encim

AdoMet

H3C-S

N

NO

H

NH2

DNA

6

5

S

AdoHcy

S

B:

encim

CH3

S encim

N

NO

CH3

NH2

DNA

6

5

vezava encima na DNA

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Gln237

Ser87

Arg165

Glu119

Phe79

Cys81

Ado Met