3/5/2011 1 LECTURE 3: LECTURE 3: CHARACTERISTICS OF CHARACTERISTICS OF ENZYME ENZYME CATALYSIS CATALYSIS Isoenzyme Isoenzyme Enzymes that perform the same catalytic Enzymes that perform the same catalytic function in different body tissues or different function in different body tissues or different organisms, but which have different organisms, but which have different sequences of amino acids in various portions sequences of amino acids in various portions of their polypeptide chain are called of their polypeptide chain are called isoenzymes isoenzymes. . Isoenzymes Isoenzymes can be separated can be separated from one another by electrophoresis from one another by electrophoresis. Proenzyme Proenzyme or or zymogen zymogen Proenzyme Proenzyme (or (or zymogen zymogen) is the name given to ) is the name given to the inactive form of an enzyme. Enzymes the inactive form of an enzyme. Enzymes (especially digestive enzymes) are often (especially digestive enzymes) are often secreted in their inactive form, transported to secreted in their inactive form, transported to the place where activity is desired, and then the place where activity is desired, and then converted to their active forms. converted to their active forms. Enzim Enzim monomerik monomerik; yang ; yang memiliki memiliki hanya hanya satu satu rantai rantai polipeptida polipeptida dimana dimana terdapat terdapat tempat tempat aktif aktif. Enzim Enzim oligomerik oligomerik; ; yang yang memiliki memiliki paling paling sedikit sedikit 2 2 dan dan sebanyak sebanyak 60 60 atau atau lebih lebih subunit yang subunit yang terikat terikat kuat kuat dalam dalam pembentukan pembentukan protein protein enzim enzim aktif aktif. Kompleks Kompleks multienzim multienzim; ; yang yang terdiri terdiri dari dari sejumlah sejumlah enzim enzim yang yang terikat terikat kuat kuat Enzyme Enzyme-Substrate Interaction Substrate Interaction Lock and Key" Hypothesis Lock and Key" Hypothesis The "Induced Fit" Hypothesis The "Induced Fit" Hypothesis "Lock and Key" Hypothesis "Lock and Key" Hypothesis Emil Fischer in 1890 proposed Emil Fischer in 1890 proposed "Lock and Key" "Lock and Key" Hypothesis Hypothesis The shape, or configuration, of the active site The shape, or configuration, of the active site is especially designed for the specific substrate is especially designed for the specific substrate involved. involved.
14
Embed
Isoenzyme LECTURE 3blog.ub.ac.id/faraca/files/2012/10/Lect3-ENZ-Charac-2011.pdf · MICHAELIS-MENTEN MODEL E = Enzyme, S = Substrate, P = Product ES = Enzyme-Substrate complex k1,
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
3/5/2011
1
LECTURE 3:LECTURE 3:CHARACTERISTICS OF CHARACTERISTICS OF
ENZYME ENZYME CATALYSISCATALYSIS
IsoenzymeIsoenzyme Enzymes that perform the same catalytic Enzymes that perform the same catalytic
function in different body tissues or different function in different body tissues or different organisms, but which have different organisms, but which have different sequences of amino acids in various portions sequences of amino acids in various portions of their polypeptide chain are called of their polypeptide chain are called isoenzymesisoenzymes. . IsoenzymesIsoenzymes can be separated can be separated from one another by electrophoresisfrom one another by electrophoresis..
ProenzymeProenzyme or or zymogenzymogen ProenzymeProenzyme (or (or zymogenzymogen) is the name given to ) is the name given to
the inactive form of an enzyme. Enzymes the inactive form of an enzyme. Enzymes (especially digestive enzymes) are often (especially digestive enzymes) are often secreted in their inactive form, transported to secreted in their inactive form, transported to the place where activity is desired, and then the place where activity is desired, and then converted to their active forms.converted to their active forms.
EnzimEnzim monomerikmonomerik; yang ; yang memilikimemiliki hanyahanyasatusatu rantairantai polipeptidapolipeptida dimanadimana terdapatterdapattempattempat aktifaktif..
EnzimEnzim oligomerikoligomerik; ; yang yang memilikimemiliki paling paling sedikitsedikit 2 2 dandan sebanyaksebanyak 60 60 atauatau lebihlebihsubunit yang subunit yang terikatterikat kuatkuat dalamdalampembentukanpembentukan protein protein enzimenzim aktifaktif..
KompleksKompleks multienzimmultienzim; ; yang yang terdiriterdiri daridarisejumlahsejumlah enzimenzim yang yang terikatterikat kuatkuat
EnzymeEnzyme--Substrate InteractionSubstrate Interaction Lock and Key" HypothesisLock and Key" Hypothesis The "Induced Fit" Hypothesis The "Induced Fit" Hypothesis
"Lock and Key" Hypothesis"Lock and Key" Hypothesis Emil Fischer in 1890 proposed Emil Fischer in 1890 proposed "Lock and Key" "Lock and Key"
Hypothesis Hypothesis The shape, or configuration, of the active site The shape, or configuration, of the active site
is especially designed for the specific substrate is especially designed for the specific substrate involved.involved.
3/5/2011
2
Because the configuration is determined by Because the configuration is determined by the amino acid sequence of the enzyme, the the amino acid sequence of the enzyme, the native configuration of the entire enzyme native configuration of the entire enzyme molecule must be intact for the active site to molecule must be intact for the active site to have the correct configuration. In such a case, have the correct configuration. In such a case, the substrate then fits into the active site of the substrate then fits into the active site of the enzyme in much the same way as a key the enzyme in much the same way as a key fits into a lock.fits into a lock.
Lock & Key ModelLock & Key Model
E n z i m S u b stra t
The "Induced Fit" Hypothesis The "Induced Fit" Hypothesis Enzymes are highly flexible, Enzymes are highly flexible, conformationallyconformationally
dynamic molecules, and many of their dynamic molecules, and many of their remarkable properties, including substrate remarkable properties, including substrate binding and catalysis, are due to their binding and catalysis, are due to their structural pliancy. structural pliancy.
Realization of the conformational flexibility of Realization of the conformational flexibility of proteins led Daniel proteins led Daniel KoshlandKoshland to hypothesize to hypothesize that the binding of a substrate (S) by an that the binding of a substrate (S) by an enzyme is an interactive process. That is, the enzyme is an interactive process. That is, the shape of the enzyme's active site is actually shape of the enzyme's active site is actually modified upon binding S, in a process of modified upon binding S, in a process of dynamic recognition between enzyme and dynamic recognition between enzyme and substrate aptly called induced fit. substrate aptly called induced fit.
In essence, substrate binding alters the In essence, substrate binding alters the conformation of the protein, so that the protein conformation of the protein, so that the protein and the substrate "fit" each other more and the substrate "fit" each other more precisely. The process is truly interactive in precisely. The process is truly interactive in that the conformation of the substrate also that the conformation of the substrate also changes as it adapts to the conformation of the changes as it adapts to the conformation of the enzyme. enzyme.
Enzim Substrat
3/5/2011
3
Induced Fit Model
Enzyme KineticsEnzyme Kinetics Enzymes follow zero order kinetics when Enzymes follow zero order kinetics when
substrate concentrations are high. Zero order substrate concentrations are high. Zero order means there is no increase in the rate of the means there is no increase in the rate of the reaction when more substrate is added.reaction when more substrate is added.
Given the following breakdown of sucrose to Given the following breakdown of sucrose to glucose and fructoseglucose and fructose
Sucrose + H20 → Glucose + Fructose
O
H
HO
H
HO
H
OH
OHHH
OH OH
HO H
H OH
O
HH
HO
H
H
H
OH
ReaksiReaksi bersifatbersifat dapatdapatbalikbalik yaituyaitu sebagiansebagiansenyawasenyawa dapatdapatdisintesisdisintesis kembalikembali daridarizatzat yang yang terdapatterdapatdalamdalam reaksireaksi
HipotesisnyaHipotesisnya adalahadalah bahwabahwaEnzimEnzim (E), yang (E), yang bertindakbertindak sebagaisebagaireaktanreaktan tapitapi tidaktidak digunakandigunakan dalamdalamreaksireaksi, , menyatumenyatu dengandengan substratsubstrat (S) (S) dalamdalam suatusuatu komplekskompleks ES ES dalamdalampembentukanpembentukan produkproduk
MICHAELISMICHAELIS--MENTEN MODELMENTEN MODEL
E = Enzyme, S = Substrate, P = ProductES = Enzyme-Substrate complex k1, k2, k3 & k4 = rate constants
3
k
1
kESE+S E+Pk
2
k
4
When the substrate concentration becomes large enough to force the equilibrium to form completely all ES the second step in the reaction becomes rate limiting because no more ES can be made and the enzyme-substrate complex is at its maximum value.
ESP2k
dtdv
[ES] is the difference between the rates of ES formation minus the rates of its disappearance.
ESESSEES211 kkk
dtd
1
3/5/2011
5
Assumption of equilibrium
k-1>>k2 (k2>>k3) the formation of product is so much slower than the formation of the ES complex. That we can assume:
Ks is the dissociation constant for the ES complex.
3
k
1
kESE+S E+Pk
2
k
4
]ES[]S][E[
KKK
1
2S
Assumption of steady state
Transient phase where in the course of a reaction the concentration of ES does not change
0ES
dtd
VV SK SM
max[ ][ ]
Michaelis-Menten Model
ES E E T
The Km is the substrate concentration where voequals one-half Vmax
There are a wide range of KM, Vmax , and efficiency seen in enzymes
SekarangSekarangy = 1/V ; x = 1/[S]y = 1/V ; x = 1/[S]a = 1/a = 1/VmaxVmax ; b = K; b = KMM//VmaxVmaxdapatdapat dianalisisdianalisis dengandengan y = a + y = a + bxbx
JikaJika 1/V 1/V dihubungkandihubungkan dengandengan 1/[S], 1/[S], suatusuatu garisgaris luruslurus akanakan dihasilkandihasilkanyang yang memotongmemotong sumbusumbu y y padapada1/1/VmaxVmax dandan sumbusumbu x x padapada --1/K1/KMMsertaserta membentukmembentuk sudutsudut terhadapterhadapsumbusumbu x x sebesarsebesar KKMM//VmaxVmax. .
y = V ; x = V/[S]y = V ; x = V/[S]a = Vmax ; b = a = Vmax ; b = --KKMM
dapat dianalisis dengan y = a + bx dapat dianalisis dengan y = a + bx
Jika V dihubungkan dengan V/[S], suatu garis Jika V dihubungkan dengan V/[S], suatu garis lurus akan dihasilkan yang memotong sumbu lurus akan dihasilkan yang memotong sumbu y pada Vmax dan sumbu x pada Vmax/Ky pada Vmax dan sumbu x pada Vmax/KMM
serta membentuk sudut terhadap sumbu x serta membentuk sudut terhadap sumbu x sebesar Ksebesar KMM
y = [S]/V ; x = [S]y = [S]/V ; x = [S]a = Ka = KMM/Vmax ; b = 1/Vmax/Vmax ; b = 1/Vmax
dapat dianalisis dengan y = a + bx dapat dianalisis dengan y = a + bx
Jika [S]/V dihubungkan dengan [S], suatu Jika [S]/V dihubungkan dengan [S], suatu garis lurus akan dihasilkan yang memotong garis lurus akan dihasilkan yang memotong sumbu y pada Ksumbu y pada KMM/Vmax dan sumbu x pada /Vmax dan sumbu x pada --KKMM serta membentuk sudut terhadap sumbu x serta membentuk sudut terhadap sumbu x sebesar 1/Vmax. sebesar 1/Vmax.
Persamaan Hanes-W oolf
-KM
[S]/V
[S]
K M/Vmax
3/5/2011
9
3/5/2011
10
STEPS OF MODEL DERIVATIONSTEPS OF MODEL DERIVATION
1.1. PembentukanPembentukan ES ES adalahadalah intiinti daridari hipotesishipotesis tersebuttersebut
(1)(1)
1.1. ReaksiReaksi E E dengandengan S S terjaditerjadi dengandengan kecepatankecepatan kk11
dandan menghasilkanmenghasilkan komplekskompleks ES (ES (enzimenzim--substratsubstrat))
2.2. KompleksKompleks ES ES dapatdapat berubahberubah menjadimenjadi E E dandan S S bebasbebas kembalikembali dengandengan kecepatankecepatan kk22, , atauatau menjadimenjadiE E dandan P P dengandengan kecepatankecepatan kk33..
3
k
1
kESE+S E+P k
2
k
4
STEPS OF MODEL DERIVATION
3/5/2011
11
4.4. Jika kJika k33 kk44 , maka reaksi bersifat “irreversible”, , maka reaksi bersifat “irreversible”, sehingga produk P tidak ada yang diubah kembali sehingga produk P tidak ada yang diubah kembali menjadi substrat asal dan kmenjadi substrat asal dan k4 4 dapat diabaikan. dapat diabaikan.
5.5. Suatu hal penting yang perlu diingat adalah bahwa Suatu hal penting yang perlu diingat adalah bahwa konstanta kkonstanta k11, k, k22, k, k33 dan kdan k44 proporsional dengan proporsional dengan G G aktivasi substrat dari reaksi yang bersangkutanaktivasi substrat dari reaksi yang bersangkutan
6.6. Pada [S] yang rendah, kebanyakan enzim berada Pada [S] yang rendah, kebanyakan enzim berada dalam bentuk bebas, sehingga penambahan S akan dalam bentuk bebas, sehingga penambahan S akan langsung terikat dengan E dan diubah menjadi P langsung terikat dengan E dan diubah menjadi P dengan demikian kecepatan awal proporsional dengan demikian kecepatan awal proporsional dengan peningkatan [S]dengan peningkatan [S]
7.7. PadaPada [S] yang [S] yang lebihlebih tinggitinggi, , kecepatankecepatan reaksireaksi bervariasibervariasidengandengan peningkatanpeningkatan [S] [S] karenakarena enzimenzim mulaimulai mengalamimengalamikejenuhankejenuhan
8.8. PadaPada [S] yang [S] yang tinggitinggi, , semuasemua enzimenzim dijenuhidijenuhi oleholehsubstratsubstrat dandan karenanyakarenanya beradaberada dalamdalam bentukbentuk komplekskompleksESES
10.10. Penurunan persamaan MichaelisPenurunan persamaan Michaelis--Menten Menten tergantung pada asumsi yang disebut tergantung pada asumsi yang disebut ”Briggs”Briggs--Haldane SteadyHaldane Steady--State”State”
11.11. Keadaan "steady state" adalah suatu Keadaan "steady state" adalah suatu keadaan dimana konsentrasi intermediat keadaan dimana konsentrasi intermediat (perantara) ES tetap konstan, sementara (perantara) ES tetap konstan, sementara konsentrasi substrat dan produk berubahkonsentrasi substrat dan produk berubah
12.12. Keadaan demikian terjadi apabila Keadaan demikian terjadi apabila kecepatan pembentukan ES sama dengan kecepatan pembentukan ES sama dengan kecepatan peruraian ESkecepatan peruraian ES
21.21. JikaJika KKMM, yang , yang merupakanmerupakan ukuranukuran affinitasaffinitasenzimenzim akanakan substratsubstrat, , disubsitusikandisubsitusikan kedalamkedalamperspers (8), (8), makamaka
(10)(10)
])S/[K(1]E[]ES[
M0
3/5/2011
13
22.22. Kecepatan reaksi katalisis dapat dinyatakan dengan Kecepatan reaksi katalisis dapat dinyatakan dengan jumlah produk yang tebentuk per satuan waktu yaitu jumlah produk yang tebentuk per satuan waktu yaitu produk dari konsentrasi kompleks ES dengan produk dari konsentrasi kompleks ES dengan kapasitas katalisis enzim kkapasitas katalisis enzim k3 3 (turnover number(turnover number).).
(11)(11)
23.23. Subsitusi [ES] dari pers. (11) kedalam pers (10) Subsitusi [ES] dari pers. (11) kedalam pers (10) memberikanmemberikan
(12)(12)
]ES[kt
]P[V 3
])S/[K(1]E[k
VM
03
24.24. PadaPada keadaankeadaan E E dijenuhidijenuhi S yang S yang berartiberarti semuasemuaenzimenzim terikatterikat dengandengan substratsubstrat dalamdalam komplekskompleks ES, ES, makamaka V = V = VmaxVmax = k= k33[E][E]00. . KemudianKemudian persamaanpersamaandiatasdiatas dapatdapat ditulisditulis dalamdalam bentukbentuk berikutberikut..
atauatau (13)(13)
25.25. PersamaanPersamaan terakhirterakhir iniini ad.ad. persamaanpersamaan MichaelisMichaelis--MentenMenten yang yang secarasecara luasluas digunakandigunakan utkutk analisisanalisisreaksireaksi enzimenzim..
VVK SM
max
( /[ ])1V
V SK SM
max[ ][ ]
26.26. StoikiometriStoikiometri perspers (13) (13) adalahadalah SS P P yaituyaitu satusatu substratsubstratdandan satusatu produkproduk ((uniuni--uniuni), ), sementarasementara banyakbanyak reaksireaksiyang yang dikatalisisdikatalisis enzimenzim melibatkanmelibatkan stoikiometristoikiometri yang yang lebihlebih komplekskompleks sepertiseperti berikutberikut;;
27.27. UntungnyaUntungnya, , persamaanpersamaan MichaelisMichaelis--MentenMenten kirakira--kirakiraberlakuberlaku untukuntuk reaksireaksi yang yang lebihlebih komplekskompleks sekalipunsekalipundengandengan mekanismemekanisme yang yang berbedaberbeda. .
S P1 + P2 (Ui-Bi) S1 + S2 P (Bi-Uni) S1 + S2 P1 + P2 (Bi-Bi)