PROTEAZE PROTEAZE TRIOZA FOSFAT-IZOMERAZA LAKTAT DEHIDROGENAZA DNA-METIL TRANSFERAZA. tripsinogen. enteropeptidaza autokataliza.
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1 PRTEAZE TRIZA FSFAT-IZMERAZA LAKTAT DEIDRGEAZA DA-METIL TRASFERAZA PRTEAZE SERISKE PRTEAZE KIMTRIPSI tripsinogen enteropeptidaza autokataliza tripsin aktivacija kimotripsinogena aktivacija proelastaze aktivacija prokarboksipeptidaze 1
2 is57 Asp Zakaj je tako reaktiven? 2. Kako se S veže na E? 3. Različna specifičnost. 4. Aktivacija zimogena. 2
3 Asp102 2 is Asp102 2 is nm 0.30nm Ser 214 Gly X -formil triptofan 3 3 Gly Gly226 Val Thr226 l 2 l l 2 l Ser189 Ser189 kimotripsin Gly Gly216 elastaza - l 2 l Asp189 tripsin 3
4 superimpozicija kimotripsinogen/kimotripsin Asp102 is157 Asp194 Asp194 Ile16 Ile16 Asp102 Gly226 Gly226 Gly216 Gly216 Ser189 is157 Ser189 4
5 3 3 P F 3 3 DFP disopropilfluorofosfat S F APMSF (4-amidino-fenil)-metan-sulfonil fluorid 5
6 3 3 3 S mesto hidrolize S S l 2 ( 2) 4 2 l substrat TPK tozil fenilalanil klormetil keton inaktivira kimotripsin ne inaktivira tripsin TLK tozil lizil klormetil keton inaktivira tripsin ne inaktivira kimotripsin 2 2 Lys 2 14 Ac 2 Ac Ac Lys 2 14 acetiliran kimotripsinogen aktivacija tripsin Ac Ac Lys 2 14 acetiliran δ kimotripsinogen *Ac 2 *Ac Ac Lys 2 14 Asp102 is57 Asp102 is57 Asp102 is57 Asp102 is57 Asp102 is57 Asp102 Asp102 Asp102 is57 is57 is57 6
7 pozitivni naboj.. X + Y- nukleofilna skupina donor protona METALPRTEAZE KARBKSIPEPTIDAZA A is69 Glu72 Zn 2+ is196 Arg145 Tyr248 Glu270 7
8 Glu270 R n - R n+1 Glu270 R n - - R n+1 Zn 2+ Zn Rn R + n+1 R n + R n+1 Glu270 - Zn 2+ Glu270 - Zn 2+ ISTEISKE PRTEAZE KATEPSI K ys25 is162 acilacija deacilacija 8
9 ASPARTATE PRTEAZE KATEPSI D Asp33 Asp231 substrati fluorogeni substrati peptidil naftilamidni metilkumarinski EDAS-DABIYL edans=(5-[(2 aminoetil)-amino] naftalensulfonska kislina dabcyl =4-[[(4 -(dimetilamino)] fenil]azo] benzojeva kislina kromogeni substrati 2 ( 2) 3 peptidil naftilamidni para-nitroanilidni 2 ( 2) 3 2 arginil benziloksikarbonilna sk. Z-Arg-MA benzoilna sk. 7-amino-4-metilkumarin 3 p-nitroanilid 2 Bz-Arg-pA (L-BAPA) 9
10 inhibitorji aminopeptidaze (amastatin, bestatin) cisteinske in serinske proteaze (antipain) serinske proteaze (APMSF, benzamidin, kimostatin, DFP, DI, TPK, TLK) aspartatne proteaze (pepstatin) amastatin bestatin ( 2) 3 2 ( 2) 3 antipain
11 2 benzamidin APMSF amidino-fenil-metana-sulfonil fluorid S F 3 3 P F 3 3 DFP disopropilfluorofosfat l l DI dikloroizokumarin kimostatin pepstatin TRIZA FSAT-IZMERAZA 11
12 trioza fosfat-izomeraza 2 2 P P 3 2- G3P D-gliceraldehid 3-fosfat DAP dihidroksi aceton fosfat [ DAP] [ G3P] K= =367 Glu165 Asn14 Lys12 is95 Asn78 Asn78 Lys12 Asn14 is95 Glu165 is95 Glu165 Lys12 12
13 Glu165 zanka zanka is95 Glu165 PGA Lys12 B + B: 2-2 P 3 2 P 3 2- izmenjava z 3 2 cis-enediol 3 B 3 + B: 2-2 P P 3 is195 is195 DAP Glu Glu165 2 P Lys12 cis enediol intermediat P Lys12 G3P is195 is195 Glu165 2 P Lys12 Glu165 2 P Lys12 13
14 LAKTAT-DEIDRGEAZA 3 3 laktat dehidrogenaza + AD + + AD laktat piruvat E+AD E E E E E+AD + AD +laktat AD AD +piruvat AD + + -laktat laktat piruvat -piruvat AD / mol aktvnega mesta ms 14
15 Tyr85 Val27, Val52 Val54, Ile96 Ala98, Ile119 Ile123 2 Asp53 Val32 Val247 Val32 Gly28 Thr97 Met55 Pro139 2 P P 2 Asp30 Lys58 Lys58 Arg101 Ala98 Glu140 Lys250 2 Glu140 Arg109 is195 S Arg171 zanka Ala98-Phe120 AD + AD 2 2 is195 3 : laktat 2 ( 2 ) 3 Arg ( 2 ) 3 Arg171 15
16 DA METIL-TRASFERAZA proteini, ki se vežejo na DA 1. 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-sheet proteins) 6. proteini z motivom β-lasnice in trakom (β-hairpin/ribbon proteins) 7. drugo 8. encimi hai DA-metiltransferaza velika domena 1-93, vmesna povezava mala domena
17 Ado Met (S-adenozil-L-metionin) domena za prepoznavanje tarče hipervariabilna regija (target recognition domain) mehanizem reakcije AdoMet Adocy S 2 S DA 6 S encim DA 6 S-encim DA 6 B: S encim DA 6 karbanionski intermediat vezava encima na DA 17
18 Phe79 Glu119 Ado Met ys81 Arg165 Ser87 Gln237 18
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