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Enzyme Kinetics-1

Enzymes are biologic polymers that catalyze the chemical reaction. They increase the rate of chemical reactions taking place within living cells without themselves suffering any overall change. The reactants of enzymecatalyzed reactions are termed substrates and each enzyme is quite specific in character, acting on a particular substrate or substrates to produce a particular products or products. Enzyme terminology All enzymes are proteins. However, without the presence of a non-protein component called a cofactor, many enzyme proteins lack catalytic activity. When this is the case, the inactive protein component of an enzyme is termed the apoenzyme, and the active enzyme, including cofactor, the holoenzyme. The cofactor may be an organic molecule, when it is known as a coenzyme, or it may be a metal ion. Some enzymes bind cofactors more tightly than others. When a cofactor is bound so tightly that it is difficult to remove without damaging the enzyme it is sometimes called a prosthetic group. Organic molecule
Inactive Protein (Apoenzyme) + cofactor

Enzyme

(Holoenzyme)
Active Protein

(Coenzyme) Metal ion

Prosthetic groups are distinguished by their tight, stable incorporation into a proteins structure by covalent or noncovalent forces. Examples include pyridoxal phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamin pyrophosphate, biotin, and the metal ions of Co, Cu, Mg, Mn, and Zn. Metals are the most common prosthetic groups. The roughly one-third of all enzymes that contain tightly bound metal ions are termed metalloenzymes. Metal ions that participate in redox reactions generally are complexed to prosthetic groups such as heme or iron-sulfur clusters. Metals also may facilitate the binding and orientation of substrates, the formation of covalent bonds with reaction intermediates (Co 2+ in coenzyme B12), or interact with substrates to render them more electrophilic (electron-poor) or nucleophilic (electron-rich). Cofactors serve functions similar to those of prosthetic groups but bind in a transient, dissociable manner either to the enzyme or to a substrate such as ATP. Unlike the stably associated prosthetic groups, cofactors therefore

must be present in the medium surrounding the enzyme for catalysis to occur. The most common cofactors also are metal ions. Enzymes that require a metal ion cofactor are termed metal-activated enzymes to distinguish them from the metalloenzymes for which metal ions serve as prosthetic groups. Coenzymes serve as recyclable shuttles-or group transfer agents-that transport many substrates from their point of utilization. Association with the coenzyme also stabilized substrates such as hydrogen atoms or hydride ions that are unstable in the aqueous environment of the cell. Other chemical moieties transported by coenzymes include methyl groups (folates), acyl groups (coenzyme A), and oligosaccharides (dolichol). Enzymes are classified by reaction type The commonly used names for most enzymes describe the type of reaction catalyzed, followed by the suffix-ase. For example, dehydrogenases remove hydrogen atoms, proteases hydrolyze proteins, and isomerase catalyze rearrangements in configuration. Modifiers may precede the name to indicate the substrate (xanthine oxidase), the source of the enzyme (pancreatic ribonuclease), its regulation (hormone-sensitive lipase), or a feature of its mechanism of action (cysteine protease). Alphanumeric designators where needed are added to identify multiple forms of an enzyme (eg, RNA polymerase III, protein kinase C). To address ambiquities, the International Union of Biochemists (IUB) developed an unambiguous system of enzyme nomenclature in which each enzyme has a unique name and code number that identify the type of reaction catalyzed and the substrates involved. Enzymes are grouped into six classes: 1. Oxidoreductases (catalyze oxidations and reductions) 2. Transferases (catalyze transfer of moieties such as glycosyl, methyl, or phosphoryl groups). 3. Hydrolases reactions ( transfer of function groups to water). 4. Lyases (addition of groups to double bonds, or formation of double bonds by removal of groups). 5. Isomerases (ransfer of groups within molecules to yield isomeric forms). 6. Ligases (catalyze the joining together of two molecules coupled to the hydrolysis of ATP).

Catalysis Occurs At the Active Site a. Lock and Key model Enzymes are very specific, and it was suggested by Emil Fischer in 1894 that this was because both the enzyme and the substrate possess specific complementary geometric shapes that fit exactly into one another. This is often referred to as "the lock and key" model. However, while this model explains enzyme specificity, it fails to explain the stabilization of the transition state that enzymes achieve. b. Induced fit model In 1958 Daniel Koshland suggested a modification to the lock and key model: since enzymes are rather flexible structures, the active site is continually reshaped by interactions with the substrate as the substrate interacts with the enzyme. As a result, the substrate does not simply bind to a rigid active site, the amino acid side chains which make up the active site are moulded into the precise positions that enable the enzyme to perform its catalytic function. In some cases, such as glycosidases, the substrate molecule also changes shape slightly as it enters the active site. The active site continues to change until the substrate is completely bound, at which point the final shape and charge is determined.The extreme substrate specificity and high catalytic efficiency of enzymes reflect the existence of an environment that is exquisitely tailored to a single reaction. Termed the active site, this environment generally takes the form of a cleft or pocket. The active sites of multimeric enzymes often are located at the interface between subunits and recruit residues from more than one monomer. The three-dimensional active site both shields substrates from solvent and facilitates catalysis. Substrates bind to the active site at a region complementary to a portion of the ligand that will not undergo chemical change during the course of the reaction. This simultaneously aligns those portions of the substrate that will undergo change with the functional groups of peptidyl aminoacyl residues. The active site also binds and orients cofactors or prosthetic groups. Many active acyl residues drawn from diverse portions of the polypeptide chain contribute to the extensive size and three-dimensional character of the active site.

Diagrams to show the induced fit hypothesis of enzyme action.

Enzyme kinetics Enzyme kinetics is the field of biochemistry concerned with the quantitative measurement of the rates of enzyme-catalyzed reactions and the systematic study of factors that affect these rates. A complete, balanced set of enzyme activities is of fundamental importance for maintaining homeostasis. An understanding of enzyme kinetics thus is important to understanding how physiologic stresses such as anoxia, metabolic acidosis or alkalosis, toxins, and pharmacologic agents affect that balance. Kinetic analysis can reveal the number and order of the individual steps by which enzymes transform substrates into products. Together with site-directed mutagenesis and other techniques that probe protein structure, kinetic analyses can also reveal details of the catalytic mechanism of a given enzyme. The involvement of enzymes in virtually all-physiological processes makes them the targets of choice for drugs that cure or ameliorate human disease. Applied enzyme kinetics therefore represents the principal way by which scientists identify and characterize therapeutic agents that selectively inhibit the rates of specific enzyme-catalyzed processes. Enzyme kinetics thus plays a central and critical role in drug discovery, comparative pharmacodynamics, and determining the mode of action of drugs.

Mechanism for a single substrate enzyme catalyzed reaction. The enzyme (E) binds a substrate (S) and produces a product (P).

Enzyme kinetics is the investigation of how enzymes bind substrates and turn them into products. The rate data used in kinetic analyses are obtained from enzyme assays.

In 1902, Victor Henri proposed a quantitative theory of enzyme kinetics, but his experimental data were not useful because the significance of the hydrogen ion concentration was not yet appreciated. After Peter Lauritz Srensen had defined the logarithmic pH-scale and introduced the concept of buffering in 1909, the German chemist Leonor Michaelis and his Canadian postdoc Maud Leonora Menten repeated Henri's experiments and confirmed his equation which is referred to as Henri-Michaelis-Menten kinetics (sometimes also Michaelis-Menten kinetics).Their work was further developed by G. E. Briggs and J. B. S. Haldane, who derived kinetic equations that are still widely used today. The major contribution of Henri was to think of enzyme reactions in two stages. In the first, the substrate binds reversibly to the enzyme, forming the enzyme-substrate complex. This is sometimes called the Michaelis complex. The enzyme then catalyzes the chemical step in the reaction and releases the product.

Saturation curve for an enzyme reaction showing the relation between the substrate concentration (S) and rate (v).

Enzymes can catalyze up to several million reactions per second. For example, the reaction catalyzed by orotidine 5'-phosphate decarboxylase will consume half of its substrate in 78 million years if no enzyme is present. However, when the decarboxylase is added, the same process takes just 25 milliseconds. Enzyme rates depend on solution conditions and substrate concentration. Conditions that denature the protein abolish enzyme activity, such as high temperatures, extremes of pH or high salt concentrations, while raising substrate concentration tends to increase activity. To find the maximum speed of an enzymatic reaction, the substrate concentration is increased until a constant rate of product formation is seen. This is shown in the saturation curve on the right. Saturation happens because, as substrate

concentration increases, more and more of the free enzyme is converted into the substrate-bound ES form. At the maximum velocity (Vmax) of the enzyme, all enzyme active sites are saturated with substrate, and the amount of ES complex is the same as the total amount of enzyme. However, Vmax is only one kinetic constant of enzymes. The amount of substrate needed to achieve a given rate of reaction is also important. This is given by the Michaelis-Menten constant (Km), which is the substrate concentration required for an enzyme to reach one-half its maximum velocity. Each enzyme has a characteristic Km for a given substrate, and this can show how tight the binding of the substrate is to the enzyme. Another useful constant is kcat, which is the number of substrate molecules handled by one active site per second. The efficiency of an enzyme can be expressed in terms of kcat/Km. This is also called the specificity constant and incorporates the rate constants for all steps in the reaction. Because the specificity constant reflects both affinity and catalytic ability, it is useful for comparing different enzymes against each other, or the same enzyme with different substrates. The theoretical maximum for the specificity constant is called the diffusion limit and is about 108 to 109 (M-1 s-1). At this point every collision of the enzyme with its substrate will result in catalysis, and the rate of product formation is not limited by the reaction rate but by the diffusion rate. Enzymes with this property are called catalytically perfect or kinetically perfect. Example of such enzymes are triose-phosphate isomerase, carbonic anhydrase, acetylcholinesterase, catalase, fumarase, -lactamase, and superoxide dismutase. Michaelis-Menten kinetics relies on the law of mass action, which is derived from the assumptions of free diffusion and thermodynamically-driven random collision. However, many biochemical or cellular processes deviate significantly from these conditions, because of very high concentrations, phase-separation of the enzyme/substrate/product, or one or twodimensional molecular movement. Some enzymes operate with kinetics, which are faster than diffusion rates, which would seem to be impossible. Several mechanisms have been invoked to explain this phenomenon. Some proteins are believed to accelerate catalysis by drawing their substrate in and pre-orienting them by using dipolar electric fields. Other models invoke a quantum-mechanical tunneling explanation, whereby a proton or an electron can tunnel through activation barriers, although for proton tunneling this model remains somewhat controversial. Quantum tunneling for protons has been observed in tryptamine. This suggests that enzyme catalysis may be more accurately

characterized as "through the barrier" rather than the traditional model, which requires substrates to go "over" a lowered energy barrier. Chemical Reactions are Described Using Balanced Equations A balanced chemical equation lists the initial chemical species (substrates) present and the new chemical species (products) formed for a particular chemical reaction, all in their correct proportions or stoichiometry. For example, balanced equation (1) below describes the reaction of one molecule each of substrates A and B to form one molecule each of products P and Q.

A+B

P+Q

(1)

The double arrows indicate reversibility, an intrinsic property of all chemical reactions. Thus, for reaction (1), if A and B can form P and Q, then P and Q can also form A and B. Designation of a particular reactants as a substrate or product is therefore somewhat arbitrary since the products for a reaction written in one direction are the substrates for the reverse reaction. The term products is however, often used to designate the reactants whose formation is thermodynamic factors strongly favor formation of the products to which the arrow points often are represented with a single arrow as if they were irreversible: A+B P+Q (2)

Unidirectional arrows are also used to describe reactions in living cells where the products of reaction (2) are immediately consumed by a subsequent enzyme-catalyzed reaction. The rapid removal of product P or Q therefore effectively precludes occurrence of the reverse reaction, rendering equation (2) functionally irreversible under physiologic conditions. Changes in Free Energy Determine the Direction & Equilibrium State of Chemical Reactions (Thermodynamics of chemical Reaction) The Gibbs free energy change G (also called either the free energy or Gibbs energy), describe both direction in which a chemical reaction will tend to proceed and the concentrations of reactants and products that will be present at equilibrium. G for a chemical reaction equals the sum of the free

energies of formation of the reaction products Gp minus the sum of the free energies of formation of the substrates Gs. G0 denotes the change in free energy that accompanies transition from the standard state, one molar concentrations of substrates and products, to equilibrium . A more useful biochemical term is G0, which defines G0 at a standard state of 10-7 M protons, pH 7.0. If the free energy of formation of the products is lower than that of the substrates, the signs of G0 and G0 will be negative, indicating that the reaction as written is favored in the direction left to right. Such reactions are referred to as spontaneous. The sign and magnitude of the free energy change determine how far the reaction will proceed. G0 = RT In keq (3) The above equation illustrates the relationship between the equilibrium constant keq and G0 , where R is the gas constant (1.98 cal/mol/K or 8.31 J/mol/K) and T is the absolute temperature in degrees Kelvin. keq is equal to the product of the concentrations of the reaction products, each raised to the power of their stoichiometry, divided by the product of the substrates, each raised to the power of their stoichiometry. For the reaction A + B P+Q [P] [Q] (4)

keq =
[A] [B] G0 may be calculated from equation (3) if the concentrations of substrates and products present at equilibrium are known. If G0 is a negative number, keq will be greater than unity and the concentration of products at equilibrium will exceed that of the substrates. If G0 is positive, keq will be less than unity and the formation of substrates will be favored. It is to be noted that since G0 is a function exclusively of the initial and final states of the reacting species, it can provide information only about the direction and equilibrium state of the reaction. G0 is independent of the mechanism of the reaction and therefore provides no information concerning rates of reactions. The Rates of Reactions Are Determined By Their Activation Energy The function of a catalyst is to increase the rate of a reaction. Catalysis do not affect reaction equilibria. Any reaction, such as S P, can be described

by a reaction coordinate diagram (Fig1). In the coordinate diagram, the free energy of the system is plotted against the progress of the reaction (reaction coordinate). In its normal stable form or ground state, any molecule (such as S or P) contains a characteristic amount of free energy. The equilibrium between S and P reflects the difference in the free energy of the ground states. In the example shown in Fig1, the free energy of their ground state of P is lower than that of S, so G o for the reaction is negative and the equilibrium favours P. This equilibrium is not affected by any catalyst.

Fig 1: Reaction coordinate diagram for a chemical reaction. The free energy of the system plotted against the progress of the reaction. This diagram is a description of the energetic course of the reaction, and the horizontal axis (reaction coordinate) reflects the progressive chemical changes (e.g., bond breakage or formation) as S is converted to P. The S and P symbols mark the free energies of the substrate and product ground states. The transition state is indicated by the symbol . The activation energies, G, for the S P and P S reactions are indicated. G o is the over-all standard free energy change in going from S to P.

A favorable equilibrium, however, does not mean that the S P conversion is fast and occurs at detectable rate. The rate of a reaction is dependent on an entirely different parameter. There is an energetic barrier between S and P that represents the energy required for alignment of reacting groups, formation of transient unstable charges, bond rearrangements, and other transformations required for the reaction to occur in either direction. This is illustrated by the energetic hill in Fig 2. To undergo reaction, the molecules must overcome this barrier and therefore must be raised to a higher energy level. At the top of the energy hill is a point at which decay to the S or P state is equally probable (it is downhill either way). This is called the transition state.

Fig 2: Reaction coordinate diagram comparing the enzyme-catalyzed and uncatalyzed reaction S P. The ES and EP intermediates occupy minima in the energetic progress curve of the enzyme catalyzed reaction. The terms G uncat and Gcat corresponds to the activation energies for the uncatalyzed and catalyzed reactions, respectively. The activation energy for the overall process is lower when the enzyme catalyzes the reaction.

Reaction Proceed via Transition States The concept of the transition state is fundamental to understanding the chemical and thermodynamic basis of catalysis. Following equation depicts a displacement reaction in which an entering group E displaces a leaving group L, attached initially to R. E + R-L E-R + L Midway through the displacement, the bond between R and L has weakened but has not yet been completely severed, and the new bond between E and R is as yet incompletely formed. This transient intermediatein which neither free substrate nor product existsis termed the transition state, E--R---L. Dotted lines represent the partial bonds that are undergoing formation and rupture. The above reaction can be thought of as consisting of two partial reactions, the first corresponding to the formation (F) and the second to the subsequent decay (D) of the transition state intermediate. As for all reactions, characteristics changes in free energy, GF and GD are associated with each partial reaction. E + R-L E---R---L GF E---R---L E-R + L GD E + R-L E-R + L G = GF + GD

For the overall reaction, G is the sum of GF and GD. As for any equation of two terms, it is not possible to infer from GF or GD. Many reactions involve multiple transition states, each with an associated change in free energy. For these reaction, the overall G the number of type of transition states through which the reaction proceeds. Stated another way: overall thermodynamics tells us nothing about kinetics. GF Defines the Activation Energy Regardless of the sign or magnitude of G, G F for the overwhelming majority of chemical reactions has a positive sign. The formation of transition state intermediates therefore requires surmounting of energy barriers. For this reason, EF is often termed the activation energy, E act, the energy required to surmount a given energy barrier. The ease- and hence the frequency-with which this barrier is overcome is inversely related to E act. The thermodynamic parameters that determine how fast a reaction proceeds thus are the GF values for formation of the transition states through which the reaction proceeds. For a simple reaction, where means proportionate to, Rate e-Eact / RT The activation energy for the reaction proceeding in the opposite direction to that drawn is equal to GD. Numerous Factors Affect the Reaction Rate The Kinetic theory also called the collision theory of chemical kinetics states that for two molecules to react they must (1) approach within bond-forming distances of one another, or collide; and (2) must possess sufficient kinetic energy to overcome the energy barrier for reaching the transition state. It therefore follows that increase the frequency or energy of collision between substrates will increase the rate of the reaction in which they participate.

Temperature

Raising the temperature increases the rate of both uncatalyzed and enzymecatalyzed reaction by increasing the kinetic energy and the collision frequency of the reacting molecules. However, heat energy can also increase the kinetic energy of the enzyme to a point that exceeds the energy barrier for disrupting the noncovalent interactions that maintain the enzymes threedimensional structure. The polypeptide chain then begins to unfold, or denature, with an accompanying loss of catalytic activity. The temperature range over which it resides. Enzymes from humans generally exhibit stability at temperatures upto 45-55C. By contrast, enzymes from the thermophilic microorganisms that reside in volcanic hot springs or undersea hydrothermal vents may be stable upto or even above 100C. As illustrated in Fig 1, the total number of molecules whose kinetic energy exceeds the energy barrier Eact (vertical bar) for formation of products increases from low (A), through intermediate (B), to high (C) temperatures. Increasing the kinetic energy of molecules also increases their motion and therefore the frequency with which they collide. This combination of more frequent and more highly energetic and productive collisions increase the reaction rate. The Q10 or temperature coefficient, is the factor by which the rate of a biologic process increases for a 10C increase in temperature. For the temperatures over which enzymes are stable, the rates of most biologic processes typically double for a 10C rise in temperature (Q 10 = 2). Changes in the rates of enzyme-catalyzed reactions that accompany a rise or fall in body temperature constitute a prominent survival feature for cold-blooded life forms such as lizards or fish, whose body temperatures are dictated by the external environment. However, for mammals and other homeothermic organisms, changes in enzyme reaction rates with temperature assume physiologic importance only in circumstances such as fever or hypothermia. Reactant Concentration The frequency with which molecules collide is directly proportionate to their concentrations. For two different molecules A and B, the frequency with which they collide will double if the concentration of either A or B are doubled. If the concentrations of both A and B are doubled, the probability of collision will increase fourfold. For a chemical reaction proceeding at constant temperature that involves one molecule each of A and B, A+BP

the number of molecules that possess kinetic energy sufficient to overcome the activation energy barrier will be a constant. The number of collisions with sufficient energy to produce product P therefore will be directly proportionate to the number of collision between A and B and thus to their molar concentrations, denoted by square brackets. Rate [A] [B] Similarly, for the reaction represented by A+2B P which can also be written as A+B+B P The corresponding rate expression is Rate [A] [B] [B] or Rate [A] [B]2 For the general case when n molecules of A react with m molecules of B, nA + mB P the rate expression is Rate [A]n [B]m Replacing the proportionality constant with an equals sign by introducing a proportionality or rate constant k characteristic of the reaction under study gives following equations Rate1 = k1[A]n[B]m Rate-1 = k-1[P] Keq Is a Ratio of Rate Constants

While all chemical reactions are to some extent reversible, at equilibrium the overall concentrations of reactants and products remain constant. At equilibrium, the rate of conversion of substrates to products therefore equals the rate at which products are converted to substrates. Rate1 = Rate-1 Therefore, k1[A]n[B]m = k-1[P] and k1/k-1 = [P] / [A]n[B]m The ratio of k1 to k-1 is termed the equilibrium constant, keq. The following important properties of a system at equilibrium must be kept in mind: (1) The equilibrium constant is a ratio of the reaction rate constants. (2) At equilibrium, the reaction rates of the forward and back reactions are equal. (3) Equilibrium is a dynamic state. Although there is no net change in the concentration of substrates or products, individual substrate and product molecules are continually being inter-converted. (4) The numeric value of the equilibrium constant Keq can be calculated either from the concentrations of substrates and products at equilibrium or from the ratio k1/k-1. Hydrogen Ion Concentration The rate of almost all enzyme-catalyzed reaction exhibits a significant dependence on hydrogen ion concentration. Most intracellular enzymes exhibit optimal activity at a pH values between 5 and 9. The relationship of activity to hydrogen ion concentration reflects the balance between enzyme denaturation at high or low pH and effects on the charged state of the enzyme, the substrates, or both. For enzymes whose mechanism involves acid-base catalysis, the residues involved must be in the appropriate state of protonation for the reaction to proceed. The binding and recognition for substrate molecules with dissociable groups also typically involves the formation of salt bridges with the enzyme. The most common charged groups are the negative carboxylate groups and the positively charged

groups of protonated amines. Gain or loss of critical charged groups thus will adversely affect substrate binding and thus will retard or abolish catalysis.

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