Multisupstratni enzimi Tehnike merenja u enzimskoj kinetici
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Multisupstratni enzimiTehnike merenja u enzimskoj kinetici
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Multisupstratni sistemi
1. Neuredjeni sekvencijalni mehanizam
3. Theorell-Chance mehanizam (ternarni kompleks se ne nagomilava)
A je vrlo često koenzim (NAD+)
4. Ping-pong mehanizam
Transferaze
2. Uredjeni sekvencijalni mehanizam
Kompleks EAB: ternarni ili centralni kompleks
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Uredjeni sekvencijalni bi-bi mehanizam
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Koncentracija B je konstantna – rešenja kinetike sekvencijalnog uredjenog mehanizma
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Koncentracija A je konstantna – rešenja kinetike sekvencijalnog uredjenog mehanizma
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Kinetika sekvencijalnih bi-bi reakcijaLinearizacija
KA – Konstanta disocijacije kompleksa EAKm(A) – Mihaelisova konstanta enzima ka supstratu A u prisustvu zasićujućih koncentracija supstrata BKm(B) – Mihaelisova konstanta enzima ka supstratu B u prisustvu zasićujućih koncentracija supstrata A
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Kinetika sekvencijalnih bi-bi reakcija
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Neuredjeni sekvencijalni mehanizam
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KA – Konstanta disocijacije binarnog kompleksa EAKm(A) – Mihaelisova konstanta enzima ka supstratu A u prisustvu zasićujućih koncentracija supstrata B, konstanta disocijacije ternarnog kompleksaKm(B) – Mihaelisova konstanta enzima ka supstratu B u prisustvu zasićujućih koncentracija supstrata A, konstanta disocijacije ternarnog kompleksa
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Koncentracija A je konstantna – rešenja kinetike sekvencijalnog neuredjenog mehanizma
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Koncentracija B je konstantna – rešenja kinetike sekvencijalnog neuredjenog mehanizma
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Ping-pong reakcije
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Konstantna konc. B- Konstantna koncentacija A
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Kinetika ping-pong bi-bi reakcija
Km(A) – Mihaelisova konstanta enzima ka supstratu AKm(B) – Mihaelisova konstanta enzima ka supstratu B
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Karakteristični LB plot ping-pong kinetike.
Fosfoglicerat mutaza (transfer fosforil grupe)
Acetil-koenzim A:arilamin acetiltransferaza (transfer acil grupe uz formiranje aciltioenzima)
Sekundarni grafik LB metode linearizacije za ping-pong rekacije.
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Odredjivanje konstanti disocijacije za protein-ligand interakcije
Neekvilibrijumska dijaliza se koristi za brza odredjivanja, pri čemu se odredjuje brzina
difuzije kroz polupropustljivu membranu
Ekvilibrijumska dijaliza
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Ekvilibrijumska gel filtracija
Primenljiva i za enzime
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Ultracentrifugiranje
Filter testovi
Spektroskopske metode
Mikrokalorimetrija
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Stehiometrijska titracija
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Surface Plasmon Resonance BiosensorOdredjivanje konstanti disocijacije
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Merenja individualnih konstanti brzina
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Merenja individualnih konstanti brzina
Metoda stalnog protoka
Vrednosti za kcat su u opsegu 1 – 107 s-1
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Metoda zaustavljenog protoka
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Metoda zaustavljenog protoka
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Metoda prekinutog protoka
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Tehnika prekinutog protoka
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Tehnika pulsnog prekinutog protoka
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Flash fotoliza
Odgovorajući supstrati često nisu dostupni
Za enzime koji zahtevaju ATP i GTP razvijen je supstrat tzv. “caged” ATP, odnosno GTP
Time scale reda ms
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Relaksacione metodeTemperature jump
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Konstante brzina enzimskih reakcija i procesi koji odredjuju brzinu
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Enzimi koji postižu katalitičku perfekciju