LECTURE 3A: ENZYME CHARACTERISTICS
Struktur Enzim +
M
Aktivator ion logam
Protein Molekul organik Tempat aktif
Apoenzim
Substrat
Coenzim
Apoenzyme + Coenzyme = Holoenzyme Bagian protein (tidak aktif)
Grup prostetik (tidak aktif)
Enzim lengkap (aktif)
Apoenzyme
the protein part of the enzyme molecule – Struktur protein enzim sangat kompleks dan berfungsi menyediakan lingkungan untuk kelangsungan reaksi dengan mekanisme tertentu – Fungsi lain adalah sebagai tempat patron (template) substrat, dan karenanya protein enzim berfungsi mengenal substrat yang dipertimbangkan menjadi dasar spesifitas enzim
Cofactor
the additional chemical groups appearing in those enzymes that are conjugated proteins.
Cofactor These cofactors are required for enzyme activity and may consist of metal ions or complex organic molecules. Some enzymes require both types of cofactors. Cofactors can be divided into three groups (i) Prosthetic group (ii) coenzyme (iii) Metal Activators
Prosthetic group – In the enzyme molecule, the cofactor may be weakly attached to the apoenzyme, or it may be tightly bound to the protein. If it is tightly bound, the cofactor is called a prosthetic group – Example: bagian porphyrin (moiety) dari hemoprotein peroxidase dan flavin-adenine dinucleotide dalam succinic dehydrogenase
Coenzyme – When the cofactor of an enzyme is a complex organic molecule other than a protein, the cofactor is called a coenzyme – Example: NAD+, NADP+, tetrahydrofolic acid dan
thyamin pyrophosphate
• Perubahan Glucose 6-phosphate ke 6 Phosphogluconic acid lactone oleh suatu enzim dehydrogenase. – Coenzymenya adalah coenzyme II; NADP+& TPN (Triphosphopyridine nucleotide) yang disebut Zwischenferment pada awalnya. – Reaksi ini terjadi melalui dua tahap reaksi yaitu oksidasi fosfat gula (glucose 6-phosphate) dan reduksi coenzyme
Activator – When the cofactor of an enzyme is a metal ion, such as the ions of magnesium, zinc, ion or manganese), the cofactor is called an activator – Examples: K+, Mn+2, Mg+2, Ca+2 dan Zn+2 Isoenzyme – Enzymes that perform the same catalytic function in different body tissues or different organisms, but which have different sequences of amino acids in various portions of their polypeptide chain are called isoenzymes. Isoenzymes can be separated from one another by electrophoresis.
Proenzyme or zymogen
− Proenzyme (or zymogen) is the name given to the
inactive form of an enzyme. Enzymes (especially digestive enzymes) are often secreted in their inactive form, transported to the place where activity is desired, and then converted to their active forms.
Substrates − The substrate is the chemical substance or substances upon which the enzyme acts
• Enzim monomerik; yang memiliki hanya satu rantai polipeptida dimana terdapat tempat aktif. • Enzim oligomerik; yang memiliki paling sedikit 2 dan sebanyak 60 atau lebih subunit yang terikat kuat dalam pembentukan protein enzim aktif. • Kompleks multienzim; yang terdiri dari sejumlah enzim yang terikat kuat
EnzymEnzym-Substrate Interaction • Lock and Key" Hypothesis • The "Induced Fit" Hypothesis
"Lock and Key" Hypothesis Emil Fischer in 1890 proposed "Lock and Key" Hypothesis The shape, or configuration, of the active site is especially designed for the specific substrate involved.
Because the configuration is determined by the amino acid sequence of the enzyme, the native configuration of the entire enzyme molecule must be intact for the active site to have the correct configuration. In such a case, the substrate then fits into the active site of the enzyme in much the same way as a key fits into a lock.
Lock & Key Model
En z im
S u b str a t
The "Induced Fit" Hypothesis • Enzymes are highly flexible, conformationally dynamic molecules, and many of their remarkable properties, including substrate binding and catalysis, are due to their structural pliancy. • Realization of the conformational flexibility of proteins led Daniel Koshland to hypothesize that the binding of a substrate (S) by an enzyme is an interactive process. That is, the shape of the enzyme's active site is actually modified upon binding S, in a process of dynamic recognition between enzyme and substrate aptly called induced fit.
• In essence, substrate binding alters the conformation of the protein, so that the protein and the substrate "fit" each other more precisely. The process is truly interactive in that the conformation of the substrate also changes as it adapts to the conformation of the enzyme.
Enzim
Substrat
Induced Fit Model
Enzyme Kinetics • Enzymes follow zero order kinetics when substrate concentrations are high. Zero order means there is no increase in the rate of the reaction when more substrate is added. • Given the following breakdown of sucrose to glucose and fructose Sucrose + H20
Glucose + Fructose H
H
H
H OH
O HO
H
H
H
O
HO
OH
H
HO H H
OH
OH
OH OH
H HO
H
• Reaksi bersifat dapat balik yaitu sebagian senyawa dapat disintesis kembali dari zat yang terdapat dalam reaksi • Jika faktor lingkungan tetap, kecepatan pembentukan produk (kecepatan reaksi) ditentukan oleh konsentrasi enzim dan substrat V = kecepatan reaksi, [E] = konsentrasi enzim & [S] = konsentrasi substrat
Apabila [S] tetap, kecepatan reaksi me-ningkat sebanding dengan peningkatan [E]
V
V
[E] 1 2 3 4
[E]
W aktu
Gambar 6. Hubungan antara kecepatan reaksi (V) dengan konsentrasi enzim (kiri), dan dengan waktu pada [E] yang berbeda (kanan)
• Apabila konsentrasi enzim tetap dan substrat meningkat, V akan meningkat mulamula-mula dan proporsional dengan peningkatan [S], tapi pada [S] yang lebih tinggi, laju peningkatan V menurun secara perlahanperlahan-lahan hingga kemudian V hampir tidak tergantung pada [S]. V
Vmax
Gambar 2.2. Hubungan antara kecepatan reaksi (V) dengan konsentrasi substrat ([S]) pada reaksi yang dikatalisis oleh suatu enzim KM
[S]
MICHAELIS-MENTEN MODEL • Leonor Michaelis dan Maud Menten pada tahun 1913 mengusulkan suatu model untuk menjelaskan kinetik reaksi enzimatis untuk satu substrat dan satu enzim (Uni(Uni-Uni reaction)
Hipotesisnya adalah bahwa Enzim (E), yang bertindak sebagai reaktan tapi tidak digunakan dalam reaksi, menyatu dengan substrat (S) dalam suatu kompleks ES dalam pembentukan produk
E+S
k1 k2
ES
k3 k4
E+P
E = Enzyme, S = Substrate, P = Product ES = EnzymeEnzyme-Substrate complex k1, k2, k3 & k4 = rate constants
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 enzymeenzyme-substrate complex is at its maximum value.
d [P ] v= = k 2 [ES] dt
[ES] is the difference between the rates of ES formation minus the rates of its disappearance.
d [ES] = k1 [E ][S] − k −1 [ES] − k 2 [ES] dt
1
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:
E+S
k1 k2
ES
k3 k4
E+P
K 2 [E][S] KS = = K1 [ES] Ks is the dissociation constant for the ES complex.
Assumption of steady state Transient phase where in the course of a reaction the concentration of ES does not change
2
d [ES] =0 dt
[E]T = [E] + [ES] MichaelisMichaelis-Menten Model
Vmax[S] V= KM + [S]
The Km is the substrate concentration where vo equals oneone-half Vmax
There are a wide range of KM, Vmax , and efficiency seen in enzymes
But how do we analyze kinetic data?
Penetuan KM dan Vmax • Harga KM bervariasi sangat besar, tapi dari kebanyakan enzim berkisar diantara 10-1 - 10-6 M (Tabel 2.1) tergantung substrat dan lingkungan seperti suhu dan kuantitas ion • Untuk mendapatkan harga KM dan Vmax, analisis langsung persamaan diatas dapat dilakukan, tapi cara ini membutuhkan waktu yang lama, dan bantuan komputer sangat penting untuk mengoptimasi harga parameter persamaan dengan cepat.
Tabel 2.1 Parameter beberapa enzim
PENDEKATAN LAIN • Linierisasi persamaan Modifikasi persamaan ke bentuk linier sehingga dapat dianalisis dengan mudah 1. Persamaan “double“double-reciprocal” atau “Lineweaver“Lineweaver-Burk” 2. Persamaan “Eadie“Eadie-Hofstee” 3. Persamaan “Hanes“Hanes-Woolf”
Persamaan “double“double-reciprocal” atau “Lineweaver“Lineweaver-Burk” Vmax[S] V= KM + [S] • Jika ruas kiri dibalik dan demikian juga ruas kanan, maka
1 KM 1 1 = . + V Vmax [S] Vmax • Sekarang persamaan ini akan mudah dianalisis dengan metode linier sedehana
• Sekarang y = 1/V ; x = 1/[S] a = 1/Vmax ; b = KM/Vmax dapat dianalisis dengan y = a + bx • Jika 1/V dihubungkan dengan 1/[S], suatu garis lurus akan dihasilkan yang memotong sumbu y pada 1/Vmax dan sumbu x pada -1/KM serta membentuk sudut terhadap sumbu x sebesar KM/Vmax.
KM/Vmax
-1/KM 1/Vmax 1/[S]
Persamaan “Eadie-Hofstee” Vmax[S] V= KM + [S]
V ( K M + [ S ]) = V max [ S ] V [ S ] = − VK M + V max [ S ] V =
− VK M + V max [ S ] [S ]
V V = −K M + Vmax [S]
• Sekarang y = V ; x = V/[S] a = Vmax ; b = -KM dapat dianalisis dengan y = a + bx • Jika V dihubungkan dengan V/[S], suatu garis lurus akan dihasilkan yang memotong sumbu y pada Vmax dan sumbu x pada Vmax/KM serta membentuk sudut terhadap sumbu x sebesar KM
V Vmax
Persamaan Eadie-Hofstee
Vmax/KM
V/[S]
Persamaan “Hanes-Woolf” Vmax [S] V= KM + [S] V (K
M
+ [ S ]) = V max [ S ]
[S ] K M + [S ] = V V max [S ] K M = V V max
+
1 V max
.[ S ]
[S] K M 1 = .[S] + V Vmax Vmax
• Sekarang y = [S]/V ; x = [S] a = KM/Vmax ; b = 1/Vmax dapat dianalisis dengan y = a + bx • Jika [S]/V dihubungkan dengan [S], suatu garis lurus akan dihasilkan yang memotong sumbu y pada KM/Vmax dan sumbu x pada -KM serta membentuk sudut terhadap sumbu x sebesar 1/Vmax.
[S]/V
Persamaan H anes-W oolf
-K M
K M /Vmax [S]
