kinetika encimov
TRANSCRIPT
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ENCIMSKA KINETIKA
Matej Huš,november 2009
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POMEN POZNAVANJA
nudi vpogled v mehanizem delovanja encima
nudi informacije o vlogi encima v celicah in odzivu na spremembe koncentracije metabolitov
pomaga razumeti, kako lahko encim kontroliramo
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PRIDOBIVANJE PODATKOV
Iščemo hitrost tvorbe produkta ali izginevanja substrata.
Variiramo: koncentracijo substrata pH temperaturo koncentracijo ligandov
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PRIDOBIVANJE PODATKOV
D-glukoza + ATP D-glukoza 6-fosfat + ADP
Diskontinuirna metoda Vzorčenje, ustavitev reakcije, meritev
koncentracije produkta (kromatografija)Kontinuirna metoda
D-glukoza 6-fosfat + NADP+ 6-fosfoglukonat + NADPH Merjenje absorpcije NADPH pri 340 nm Sklopljena reakcija ne sme biti počasna
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PRIDOBIVANJE PODATKOV
Čistost vseh kemikalijBrez encimskih kontaminacijStabilnost encimovStabilnost pogojev (temperatura, pH)Hitrost reakcije v stacionarnem stanju
konstantna in sorazmerna koncentraciji encima
Meritev začetne hitrosti reakcije
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POTEK KATALIZIRANE REAKCIJE
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POTEK KATALIZIRANE REAKCIJE
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POTEK KATALIZIRANE REAKCIJE
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POTEK KATALIZIRANE REAKCIJE
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REAKCIJE Z ENIM SUBSTRATOM
E + S ES E + P
1. obravnava (Henri 1902, Michaelis in Menten
1913): hitra vzpostavitev ravnotežja
2. obravnava (Briggs in Haldane 1925): predpostavka stacionarnega stanja
k1
k-1
k2
maxv Sv
K S
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REAKCIJE Z ENIM SUBSTRATOM
maxv Sv
K S
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LINEARIZACIJA PODATKOV
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DIREKTNI LINEARNI GRAF
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PORAZDELITEV NAPAK
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PORAZDELITEV NAPAK
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PORAZDELITEV NAPAK
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PARAMETRI
catmax t
m
k E Sv Sv
K S K S
kcat katalitična konstanta, pretvorbeno
število (turnover number) [s-1]
Km konstanta afinitete [mol L-
1]
kcat / Km konstanta specifičnosti [M-1
s-1]
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SPECIFIČNOST HEKSOKINAZ
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IREVERZIBILNA INHIBICIJA
Inhibitor permanentno deaktivira encim.
E + I EI E’k1
k-1
k2
i
E I Ea EI IK
EI EI
i
Ea EI IEI
K
2
d
d i
V IEak EI
t K I
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REVERZIBILNA INHIBICIJA
Kompetitivna inhibicija (competitive inhibition)
Nekompetitivna inhibicija (non-competitive inhibition)
Akompetitivna inhibicija (uncompetitive inhibition)
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KOMPETITIVNA INHIBICIJA
Inhibitor se veže izključno z E v EI.
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NEKOMPETITIVNA INHIBICIJA
Inhibitor se veže z E ali ES v EI oz. EIS.
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AKOMPETITIVNA INHIBICIJA
Inhibitor se veže izključno z ES v EIS.
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AKOMPETITIVNA INHIBICIJA
Inhibitor se veže izključno z ES v EIS.
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INHIBICIJA S SUBSTRATOM
Neproduktivna vezava
Substratna inhibicija
Primer: 6-fosfofruktokinaza
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ENCIMSKA AKTIVACIJA
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VPLIV pH NA AKTIVNOST
Ena ionizirajoča skupina
Dve ionizirajoči skupini
E + H+ EH+Ka
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VPLIV pH NA AKTIVNOST
Ena ionizirajoča skupina
Dve ionizirajoči skupini
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VPLIV T NA AKTIVNOST
Pozitivna korelacija (Arrheniusova enačba)
Negativna korelacija (denaturacija)
aE
RTk Ae
E E’K
0 0
0 0 0lnH T S
RT
RT K G H T S
K e
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VPLIV T NA AKTIVNOST
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REAKCIJE Z 2 SUBSTRATOMA
Encimska substitucija (ping-pong)
Trojni kompleks nastane po urejeni poti nastane po naključju
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REAKCIJE Z 2 SUBSTRATOMA
Encimska substitucija (ping-pong)
Trojni kompleks nastane po urejeni poti nastane po naključju
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REAKCIJE Z 2 SUBSTRATOMA
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REAKCIJE Z 2 SUBSTRATOMA
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REAKCIJE Z 2 SUBSTRATOMA
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MATEMATIČNA OBDELAVA
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MATEMATIČNA OBDELAVA
Trojni kompleks - primarni graf (koncentracijo substrata B držimo konstantno)
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MATEMATIČNA OBDELAVA
Trojni kompleks - sekundarni graf(koncentracijo substrata B držimo konstantno)
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MATEMATIČNA OBDELAVA
Ping-pong mehanizem - primarni graf(koncentracijo substrata B držimo konstantno)
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MATEMATIČNA OBDELAVA
Ping-pong mehanizem - sekundarni graf(koncentracijo substrata B držimo konstantno)
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PREGLED
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UREJENI ALI NAKLJUČNI TERCIARNI KOMPLEKS