mehanizmi dejstva enzima himotripsin
TRANSCRIPT
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Mehanizmi dejstva enzima
Himotripsin
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Principi katalize
� Specifična kiselo-bazna kataliza� Elektrostatska kataliza� Elektrofilna kataliza� Nukleofilna kataliza (kovalentna kataliza)
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Nukleofilna kataliza
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Opšta kiselo-bazna kataliza
� log k2 = A + β pKa Brønsted-ova jednačina
� log k2 = A - α pKa� Jonizaciono stanje katalizatora –odredjuje i efikasnost katalize...
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Intramolekularna kataliza
� Efektivna koncentracija grupe na enzimu: slučaj opšte bazne katalize
Hidroliza aspirina – efektivna koncentracija karboksilne grupe u aspirinu je 13 M
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� Efektivna koncentracija nukleofila u intramolekularnoj katalizi
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Entropija
� Translaciona, rotaciona i unutrašnja
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Elektrostatska kataliza
� E = e1e2/Dr� Enzimi mogu da stabilizuju polarna prelazna stanja bolje od vode.� 1. Dva ili tri fiksiranja dipola u enzimu mogu da stabilizuju
naelektrisanje kao i voda.� 2. Jonski par se može stabilizovati fiksiranim dipolima u enzimu
efikasnije nego u vodi.� Enzimi koriste delove strukture da solvatizuju, a ne vodu.
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Kataliza jonima metala
� a) Elektrofilna kataliza
� Karboksipeptidaza (polarizacija amida na nukleofilni napad i stabilizacija tetrehedralnog intermedijera).
b) Izvor hidroksilnih jona na neutralnom pH (karboanhidraza)
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Kovalentna kataliza
� Elektrofilna kataliza formiranjem Šifove baze
Metilenski ugljenik se aktivira i postaje nukleofilan.
Karbonilni ugljenik se aktivira na nukleofilni napad zbog snažnog povlačenja
elektrona ka protonovanom azotu.
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Acetoacetat dekarboksilaza
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Izopropil lizin je detektovan u hidrolizatu enzima!
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Aldolaza i transaldolazaKatalizuju kondenzaciju aldola i povratnu reakciju uz formiranjeŠifove baze (ugljenik je aktiviran u enaminu)
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Piridoksal fosfat (elektrofilna kataliza)
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Uklanjanje αααα-vodonika: racemizacija ili transaminacija
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ββββ-dekarboksilacija
Izmena bočnog lanca
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Nukleofilna kataliza
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Mehanizam dejstva himotripsina
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Mehanizmi enzimimakatalizovanih reakcija
� Redosled konverzijerazličitih kompleksaenzima kojima se S konvertuje u proizvod
� Brzine kojima se ovikompleksiinterkonvertuju
� Strukture
� Kinetičke studije� X-ray kristalografija� Protein inženjering� Detekcija intermedijera� Hemijsko obeležavanje i
hemijske modifikacijebočnih ostataka u enzimima
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Proteaze
� Serin proteaze
� Karboksil – aspartil (kisele) proteaze
� Tiol (cistein) proteaze
� Cink proteaze
� Monomerni enzimi, mase 15 – 35 kDa
� Adenovirusi (cistein proteaze), pikornavirusi (serin proteaze),
retrovirusi (aspartil proteaze).
� (N-termunus) S1 – S1` veza se raskida
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Familija serin proteaza - struktura
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Serin proteaze
Dva domena, katalitička trijada Asp-102, His-57, Ser-195 se nalazi
izmedju dva domena
Formira se acil-enzim.
Nema direktnih dokaza za formiranje tetraedarskog intermedijera
Indirektni dokazi: x-ray Enz-Inh kompleksa
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Katalitička trijada u serin proteazama
Protein inženjeringom potvrdjena uloga katalitičke trijade
Katalitička trijada Asp-102, His-57, Ser-195
charge-relay systempKa Asp neobično niska (2)
His jonizuje na pH 6.8
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Doprinos oksianjon vezivnog mesta katalizi
� Nakon mutageneze katalitičke trijade (Ala), katalizaje smanjena, ali i dalje 1000 puta veća od spontanehidrolize
� Komplementarnost aktivnog mesta enzima prelaznomstanju reakcije (vibraciona spektroskopija)
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Deacilacija
Acilacija – dodatna mesta vezivanja S1 – S5 povećavaju kcat, ali ne utiču na Km
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Mehanizam dejstva himotripsina
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Serin proteaze – tetreadarskiintermedijer
Intermedijer se ne nagomilava tokom reacije
Dokazi za njegovo postojanje su samo indirektni
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Himotripsin – struktura aktivnogmesta
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Himotripsin – neproduktivnovezivanje
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Mehanizam dejstva himotripsinana amidnim supstratima
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Hemijska modifikacija His-57
TPCK – N-p-toluensulfonil-L-fenilalanin hloromehtilketonmodifikuje His-57 himotripsina, ali ne i tripsina
TLCK - N-p-toluensulfonil-L-lizin hlorometillketon modifikuje aktivnomesto tripsina, a neaktivan je na himotripsinu
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Zimogeni – aktivacija himotripsina
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Krucijalna uloga Ile-16: eksperimentdvostrukog obeležavanja
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Serin proteaze – specifičnost premasupstatima (struktura vezivnog džepa)
� Himotripsin: Ile-16 i Asp-194 formiraju vezivni džep gradeći jonski most u okruženju niske dielektrične konstante
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Odnos mehanizma dejstva
himotripsina i drugih proteinaza
� (i) nukleofil – napad na karbonilnu grupu + formiranjetetraedarskog intermedijera
� (ii) pozitivno naelektrisana vrsta (ili –NH- grupe koje grade vodoničnu vezu)– povećava susceptibinost karbonilne grupe nanukleofilni napad i stabilizuje tetraedarski intermedijer
� (iii) donor protona