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• 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
9
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
11
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
14
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
16
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
17
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
18
Gln237
Ser87
Arg165
Glu119
Phe79
Cys81
Ado Met