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KINETICS
Enzyme Action
Each enzyme has a unique three-dimensional shape that binds and recognizes a group of reacting molecules called substrates. The active site of the enzyme is a small pocket to which the substrate directly binds. Some enzymes are specific only to one substrate; others can bind more than one substrate.
Enzyme-Substrate Binding
Molecular Recognition
How does an enzyme bind a substrate, reduce the activation barrier, and produce a product?
vs.
Energy of activation: G
G Gcat
Effect of catalysis
AB
GT1
Effect of temp
GT2 (T1 > T2)
AB
Stabilization of the transition state: covalent bonds, metals, acid-base, and proximity.
E+S ES
Reduced entropy in ES formation.
E+P
Michaelis-Menten Kinetics
k1
E + S ES
k-1
k2
E+P
Assumptions: [1] Steady-state of the intermediate complex ES [2] Neglect back rxn from product (k-2; not shown) [3] Conservation of mass ([ET] = [E] + [ES])
Vmax = k2[ET]
where:
Km =
(k-1 + k2) k1
Michaelis-Menten Kinetics
Michaelis-Menten Kinetics
Many types of inhibition can be included in the MM model as well as multiple substrates and steps:
Reaction Schemes: single substrate multiple substrate single displacement double disp (ping-pong)
Zymogens
Many enzymes are active as soon as theyre made. However, some are made in an inactive form and stored. This inactive form is called a zymogen or proenzyme. To become active, the body needs only to cleave off a small peptide fragment.
Feedback Control
Some enzymes (allosteric enzymes) bind molecules called regulators (different from the substrate) that can affect the enzyme either positively or negatively
Positive regulator: speeds up the reaction by changing the shape of the active site -- substrate binds more effectively Negative regulator: slows down reaction by preventing proper substrate binding, again, by changing enzyme shape
Feedback control: the end product acts as a negative regulator. If there is enough of the end product, it will slow down the first enzyme in a pathway.
A hyperbolic curve between V0 and [S] was revealed by in vitro studies using purified enzymes
It was the initial velocity (rate), V0, that was measured, so the change of [S] could be ignored. The catalysis was assumed to occur as:
The enzyme will become saturated at high [S]: the V0 will not be affected by [S] at high [S].
Vmax is extrapolated from the plot: V0 approaches but never quite reaches Vmax.
The effect on V0 of varying [S] is measured when the enzyme concentration is held constant. Hyperbolic relationship between V0 and [S]
A mathematical relationship between V0 and [S] was established (Michaelis and Menten, 1913; Briggs and Haldane, 1925)
k1 k2
) E+S ES E+P ( Formation of ES is fast and reversible. The reverse reaction from PS (k-2 step) was assumed to be negligible. The breakdown of ES to product and free enzyme is the rate limiting step for the overall reaction. ES was assumed to be at a steady state: its concentration remains constant over time. Thus V0 = k2[ES]
k
k
1
-1
k1([Et]-[ES])[S]=k-1[ES] + k2[ES] ([Et] is the total enzyme concentration.) Km is called the Solve the equation for [ES]:
Michaelis constant.
V0 = k2[ES]
The maximum velocity is achieved when all the enzyme is saturated by substrate, i.e., when [ES] =[Et]. Thus Vmax =k2[Et]
The Michaelis-Menten Equation
The Vmax and Km values of a certain enzyme can be measured by the double reciprocal plot (i.e., the Lineweaver-Burk plot).
The Michaelis-Menten equation, but not their approximated mechanism applies to a great many enzymes
Most enzymes (except the regulatory enzymes) have been found to follow the Michaelis-Menten kinetics, but their actual mechanisms are usually more complicated (by having more intermediate steps) than the one assumed by Michaelis and Menten. The values of Vmax and Km alone provide little information about the number, rates, or chemical nature of discrete steps in the reaction.
For If k2 is rate-limiting, k2<<k-1, Km = k-1/k 1 Km equals to the dissociation constant (Kd) of the ES complex; Km represent a measure of affinity of the enzyme for its substrate in the ES complex.
Plot Eadie-Hofstee dan Hanes diturunkan dari persamaan Lineweaver-Burk dengan mengalikan kedua sisi persamaan dengan faktor vo Vmax sehingga akan diperoleh persamaan garis lurus selanjutnya dipergunakan untuk menghitung Vmax dan Km
Dengan cara penurunan yang mirip, Hanes-Woolf mengalikan perasamaan Lineweaver-Burk dengan [So] maka diperoleh:
Plot Eadie Hofstee dan Hanes banyak digunakan pada studi kinetik enzim, namun demikian studi enzim secara umum masih menggunakan plot Lineweaver Burk.
1 Km 1 1 v Vmax [S ] Vmax
Plot 1/v versus 1/[S]. Slope is Km/Vmax, intercept is 1/Vmax
Graphical Solution
intercepts
1/ V
1 Km 1 1 v Vmax [S ] Vmax
-1/ Km
1/ [S]
Example: Lineweaver-Burk
[S] x 10 M 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
-5
V, M/min x 10 1.17 1.50 1.75 1.94 2.10 2.23 2.33 2.42 2.50
-5
Resulting Plot
Michaelis-Menten Kinetics
Vmax [S ] k 2 [E 0 ][S ] v K m [S ] K m [S ]
Vmax = 1/2.8687 x 10-4 = 3.49 x 10-5 M/min Km= 0.5686 x Vm = 1.98 x 10-5 M
Other Methods
Eadie-Hofstee plot
v Vmax
Hanes- Woolf
v Km [S ]
[S ] K m v Vmax
1 [S ] Vmax
Comparison of Methods
Lineweaver-Burk: supposedly gives good estimate for Vmax, error is not symmetric about data points, low [S] values get more weight Eadie-Hofstee: less bias at low [S] Hanes-Woolf: more accurate for Vmax. When trying to fit whole cell data I dont have much luck with any of them!
PERSAMAAN HALDAN UNTUK REAKSI REVERSIBEL Reaksi enzimatis dalam sel sering berlangsung secara reversibel. Reaksi substrat tunggal, S P, berlangsung melalui pembentukan satu kompleks intermediate, arah ke kanan dianggap sebagai kompleks ES dan sebaliknya kompleks EP E+S ES/EP P+E
Persamaan MM arah kekanan pada [Eo] tetap dengan laju awal vf dan Vsmax
Persamaan MM ke arah sebaliknya pada [Eo] tetap dengan laju awal vb dan VPmax
Perumusan Haldan hubungan antara konstanta laju dan kesetimbangan reaksi pada reaksi kesetimbangan adalah
Karena
Maka:
Bila konstanta kesetimbangan diketahui, maka persamaan tersebut dapat digunakan untuk memvalidasi konstanta laju yang diperoleh Secara umum Km dari arah reaksi metabolisme penting akan sedikit lebih kecil dari arah sebaliknya. Namun arah metabolisme dipengaruhi juga oleh [S] dan [P] dalam sel
Untuk reaksi sederhana order pertama A B Maka laju reaksi pada t adalah -d[A]/dt = d[B]/dt = k[A] Integrasi dari persamaan tersebut memberikan persamaan ln[Ao] ln [A] = kt [A] = [Ao]e kt
konsentrasi A
Persamaan integrasi dari sebagian besar mekanisme reaksi akan lebih rumit . Untuk reaksi substrat tunggal yang membentuk suatu kompleks intermediate dengan konsentrasi awal dari [S] >>> [E] dinyatakan sebagai:
E+S
k1
k-1
k2
ES E+P
k-2
Laju penambahan [ES] pada waktu t (pada periode awal dimana pembentukan [P] diabaikan dinyatakan sebagai: d[S]/dt =k1 [E][S] - k-1 [ES] - k2 [ES] d[S]/dt = k1 ([Eo] - [ES])([So] [ES] [P]) - k-1 [ES] - k2 [ES] karena : [So]>>[Eo], maka ([So]-[ES]-[P] ~ [So] Jadi : d[S]/dt = k1 ([Eo] - [ES])([So] ) - k-1 [ES] - k2 [ES]
Konstr.
S
E+S
ES
EP
E+P
ES
Waktu
Bagian linier dari grafik [P] vs t menunjukkan fase steady state dari reaksi dengan slope = k2[E][So]/([So]/Km) yang didapat dari mensubstitusi (k-1 + k2)/k1 dengan Km dari persamaan integrasinya Jika bagian grafik steady state linier, ekstrapolasinya akan memotong sumbu t pada t = 1/(k1[So]+Km) dan disebut sebagai periode induksi. Kurva perubahan konsentrasi akan lebih rumit bila reaksinya seperti; E+S ES EP E+P
E+S
ES
EP
E+P
Konstr.
EP E P ES
Waktu
40,000,000 molecules of H2O2 are converted to H2O and O2 by one catalase molecule within one second!
The kinetic parameters kcat and Km are often studied and compared for different enzymes
Km often reflects the normal substrate concentration present in vivo for a certain enzyme. The catalytic efficiency of different enzymes is often compared by comparing their kcat/Km ratios (the specificity constant).
kcat/Km is an apparent second-order rate constant (with units of M-1S-1), relating the reaction rate to the concentrations of free enzyme and substrate.
The value of kcat/Km has an upper limit (for the perfected enzymes)
It can be no greater than k1. The decomposition of ES to E + P can occur no more frequently that E and S come together to form ES. The most efficient enzymes have kcat/Km values near the diffusioncontrolled limit of 108 to 109 M-1S-1.
Catalytic perfection (rate of reaction being diffusion-controlled) can be achieved by a combination of different values of kcat and Km.
kcat
catalyzed
uncatalyzed
kcat
Rate enhancement: ratio of the rates of the catalyzed and the uncatalyzed reactions.
Enzyme-catalyzed reactions of two or more substrates can also be analyzed by the Michaelis-Menten approach
Each substrate will have one characteristic Km value. Noncovalent ternary complex (with two substrates bound to the enzyme concurrently) may or may not be formed for the bisubstrate reactions depending on the mechanism. Steady-state kinetics can often help distinguish these two mechanisms.
In those enzyme-catalyzed bisubstrate reactions where a ternary complex is formed, the two substrates may either bind in a random sequence or in a specific order.
No ternary complex is formed in the Ping-Pong (or double displacement) mechanism: The first substrate is converted to a product that leaves the enzyme active site before the second substrate enters.
Maintaining the concentration of one substrate (S2) constant, the double reciprocal plots made by varying the concentration of the other substrate (S1) will not intersect.
S1
Rates of individual steps for an enzymecatalyzed reaction may be obtained by presteady state kinetics
The enzyme (of large amount) is used in substrate quantities and the events on the enzyme are directly observed. Rates of many reaction steps may be measured independently. Very rapid mixing and sampling techniques are required (the enzyme and substrate have to be brought together in milliseconds and measurements also be made within short period of time).
Rapid kinetics or pre-steadystate kineticsis applied to the observation of rates of systems that occur in very short time intervals (usually ms or sub-ms scale ) and very low product concentrations. This period covers the time from the enzyme encountering its target (either a substrate, inhibitor or some other ligands) to the point of system settling to equilibrium.
(ms or sub-ms)
specific activity is the amount of product formed by an enzyme in a given amount of time under given conditions per milligram of enzyme. The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time (mol L 1s 1) The enzyme activity is the moles converted per unit time (rate reaction volume). Enzyme activity is a measure of quantity of enzyme present. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively large unit. A more practical value is 1 enzyme unit (EU) = 1 mol min-1 ( = micro, x 10-6). The specific activity is the moles converted per unit time per unit mass of enzyme (enzyme activity / actual mass of enzyme present). The SI units are katal kg-1, but more practical units are mol mg-1 min1. Specific activity is a measure of enzyme efficiency, usually constant for a pure enzyme. If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific activity, allowing purity to be calculated. The % purity is 100% (specific activity of enzyme sample / specific activity of pure enzyme). The impure sample has lower specific activity because some of the mass is not actually enzyme.
Turnover number
The turnover number of carbonic anhydrase: Carbonic anhydrase of erythrocytes (Mr 30,000) has one of the highest turnover numbers among known enzymes, it catalyses the reversible reaction of CO2: H2O + CO2 -> H2CO3 This is an important process in the transport of CO2 from the tissues to the lungs. If 10g of pure carbonic anhydrase catalyses the hydration of 0.30g of CO2 in 1min at 37C at Vmax, and the reaction volume is 1ml. What is the turnover number (Kcat) of carbonic anhydrase expressed in units of per min and per sec)? Mr of CO2 is 44.