Enzymes are highly specific, powerful catalysts of biological systems that help to accelerate reactions taking place in organisms by factors of millions or more. Enzymes: Basic Concepts & Kinetics After interacting with this Learning Object, the learner will be able to: Describe Enzymes and their components. Recall Energetics of enzymatic reactions. List out models for enzyme-substrate binding. Define kinetics of enzymatic reactions. Define enzyme inhibition. Learning Objectives: Enzymes: Basic Concepts and Kinetics Molecular & cell Biology
Transcript
Enzymes are highly specific, powerful catalysts of biological systems that help to accelerate reactions taking place in organisms by factors of millions or more.
Enzymes: Basic Concepts & Kinetics
After interacting with this Learning Object, the learner will be able to: Describe Enzymes and their components.
Recall Energetics of enzymatic reactions.
List out models for enzyme-substrate binding.
Define kinetics of enzymatic reactions.
Define enzyme inhibition.
Learning Objectives:
Enzymes: Basic Concepts and KineticsMolecular & cell Biology
Most enzymes are made up of a protein part known as the apoenzyme as well as a cofactor which can either be an organic molecule known as a coenzyme or a metal ion. These cofactors are essential for the enzyme to be catalytically functional and the complete functional enzyme is referred to as the holoenzyme. Pyruvate dehydrogenase is a complex enzyme which uses Thiamine pyrophosphate as its coenzyme while carbonic anhydrase uses zinc ion as its cofactor.
Enzymes & their componentss
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
Enzymes are classified on the basis of the reactions that they catalyze. Most enzymes are named by adding the suffix ‘ase’ either to their substrate or the type of activity they carry out. However as more enzymes came to be known, it became increasingly difficult to name them in this manner. Classification by international organizations has therefore led to six enzyme classes with many subgroups within each class, depending upon the type of reaction being catalyzed. Every enzyme has a unique, four-part classification number known as the Enzyme Commission number (E.C. number), in which subclass number gives finer details about that particular enzyme reaction.
Enzymes & their componentss
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
Conversion of substrate to product proceeds through formation of a transition state. The free energy of activation of an uncatalyzed reaction is very high. Enzymes form favorable interactions with the substrate and facilitate formation of the transition state by lowering the free energy of activation. The transition state then dissociates to give the product and regenerates free enzyme. For a reaction to be spontaneous, the ∆G must be negative. It must be emphasized that enzymes do not alter the equilibrium of a reaction.
Energetics of enzymatic reactions
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
Fischer’s hypothesis is aptly defined as the ‘lock-and-key’ hypothesis. Any lock, which is analogous to an enzyme, can have only one suitable key of appropriate shape and size to open it. The various available keys, which are analogous to the thousands of substrates available, can attempt to open the lock but only one will be the perfect fit that is capable of opening the lock. Similarly only one particular substrate will fit into the active site of the enzyme and the enzymatic reaction can occur
Models for enzyme-substrate binding
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
According to the Fischer’s hypothesis, enzymes and their substrates possess specific complementary geometric shapes that fit exactly into each other. This model accounts for the specificity of enzymes but fails to account for stabilization of the transition state. Koshland modified this hypothesis and suggested that the active site of an enzyme gets continually reformed based on the interactions that it establishes with the substrate molecule. This accounts for both the enzyme specificity and the stabilization of the transition state since the enzyme is not considered to be a rigid molecule.
Models for enzyme-substrate binding
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
Enzymes catalyze the formation of product from its substrate via an enzyme-substrate intermediate complex. During the initial stages of the reaction, the equilibrium favors product formation rather than dissociation of the [ES] complex to give back the substrate. The number of moles of product formed per second during these stages determines the reaction velocity for that particular enzyme. Vo has an almost linear relation with substrate concentration when the substrate concentration is low but becomes independent at higher concentrations
Kinetics of enzymatic reactions
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
The Michaelis-Menten model for enzyme kinetics assumes that the breakdown of [ES] complex to give back free substrate is negligible and also assumes steady-state conditions whereby the rates of formation and breakdown of the [ES] complex are equal. The reaction velocity increases linearly with substrate concentration when [S] is low but becomes independent at higher concentrations. The maximal velocity that can be achieved by an enzyme refers to the state in which all its catalytic sites are occupied. The substrate concentration at which the reaction velocity is equal to half its maximal value is known as the Michaelis constant, Km.
Kinetics of enzymatic reactions
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
The Lineweaver-Burk equation or the double reciprocal plot is a useful tool that can be plotted using simple experimental data from kinetics experiments. This equation is derived from the Michaelis-Menten equation by taking reciprocals on both sides and then plotting a graph of 1/Vo Vs 1/[S]. The Y-intercept on this graph can be used to deduce the value of Vmax while the X-intercept gives the value of Km.
Kinetics of enzymatic reactions
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
Enzyme inhibition can either be reversible, where the inhibitor can dissociate quickly from the enzyme, or irreversible, where the inhibitor dissociates very slowly from the enzyme and can covalently modify the enzyme thereby rendering it unsuitable for further catalysis reactions. Reversible inhibition can be further classified as competitive, uncompetitive and mixed inhibition.
In competitive inhibition, the inhibitor molecule is structurally similar to the substrate and therefore binds to the enzyme at the active site. Binding of inhibitor prevents substrate from binding, thereby decreasing the reaction rate. The Vmax in this type of inhibition remains the same and only the Km is altered. Competitive inhibition can be overcome by suitably increasing the substrate concentration, which allows the substrate to out-compete the inhibitor for the enzyme’s active site.
In the case of uncompetitive inhibition, the substrate and inhibitor both have different binding sites on the enzyme . However, the inhibitor binds only to the enzyme-substrate complex and not to the enzyme alone. Binding of inhibitor to the ES complex prevents any further reaction and no product formation is observed. Both the Km and Vmax are found to decrease with this type of inhibition.
Enzyme inhibition
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
A mixed inhibitor also binds to the enzyme at a site distinct from the substrate binding site with the difference being that it can bind either to enzyme or ES complex. Binding of either one brings about conformational changes in the enzyme structure thereby affecting binding of the other. This inhibition can be reduced but not overcome by increasing substrate concentration. Both Km and Vmax are altered in this type of inhibition.
Enzyme inhibition
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
Enzymes & their components
1. Enzymes: Majority of enzymes are proteins (some are RNAs) that increase the rates of biochemical reactions in living organisms. They are highly specific molecules that catalyze only a particular group of reactions and like other catalysts, they are regenerated at the end of a reaction. The rates of enzyme catalyzed reactions are often more than million times faster than those of un-catalyzed ones. Molecular weights of enzymes range from 12,000 to more than 1 million daltons. Enzymes offer the advantages of high reaction rates, substrate specificity, capacity for regulation and mild reaction conditions when compared to any other chemical catalysts.
2. Apoenzyme: The protein part of anenzyme that requires other additional components in order to make it catalytically functional is known as the apoenzyme or apoprotein.
3. Cofactor: Many enzymes require the presence of an additional component known as a cofactor for their catalytic activity. These cofactors can either be metal ions such as Fe2+, Mg2+, Mn2+ etc. or organic molecules known as coenzymes. A coenzyme or metal ion that is very tightly or covalently bound to the apoenzyme is known as the prosthetic group.
4. Holoenzyme: The complete, catalytically functional enzyme containing both the protein part as well as its cofactor is known as the holoenzyme.
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
5. Enzyme classification: Enzymes are classified on the basis of the reactions that they catalyze. Most enzymes are named by adding the suffix ‘ase’ either to their substrate or the type of activity they carry out. Classification by international organizations has led to six enzyme classes with many subgroups within each class, depending upon the type of reaction being catalyzed. Every enzyme has a unique, four-part classification number known as the Enzyme Commission number (E.C. number)
Enzymes & their components
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
Energetics of enzymatic reactions
2. Substrate(s): The molecules present at the beginning of a reaction that are modified by means of the enzyme are known as substrates. An enzymatic reaction may have one or more substrates depending upon the reaction.
3. Product(s): The molecules produced as a result of an enzymatic reaction are known as the products. A reaction may yield one or more products with the enzyme being regenerated at the end of the reaction.
4.Transition state: Enzymatic reactions proceed through formation of a transition state i.e. an intermediate state between substrate and product having higher free energy than that of either the substrate or product. The transition state is the least stable species of the reaction pathway due to its high free energy and is therefore the most seldom occupied.
5. Free energy: The Gibbs free energy is a useful thermodynamic property that can be used to understand enzymatic reaction mechanisms. The free energy difference between the reactants and products as well as the energy.required to activate conversion of reactants to products can provide information about the spontaneity and rate of a reaction. The free energy change (∆G) of a reaction is only dependent on the free energy of the reactants and products and not on the path of the reaction.
Molecular & cell Biology
1. Enzyme: The biocatalyst responsible for bringing about an increase in the rate of reaction for conversion of substrate to product.
Enzymes: Basic Concepts and Kinetics
Energetics of enzymatic reactions
A reaction can occur spontaneously only if the ∆G is negative.
6. Free energy of activation (∆G‡): The rate of any reaction is dependent on the Gibbs free energy of activation (∆G‡) i.e. the energy difference between the substrate and the transition state. Enzymes function to lower this energy gap by forming favourable interactions with the substrate, thereby facilitating formation of the transition state and allowing a larger number of molecules to overcome this energy barrier. Enzymes do not function by modifying the reaction equilibrium but only alter the reaction rate.
2. Koshland’s induced fit model: Daniel Koshland in 1958 suggested that the active site of an enzyme gets continually reformed and reshaped based on the interactions that it forms with the substrate molecule. Therefore, rather than assuming a rigid shape, the enzyme’s active site gets molded.
into the exact shape and position required to carry out catalysis of the substrate. This accounts for both the enzyme specificity and the stabilization of the transition state.
3. Active site: All enzymes possess an active site that is lined with around 4-5 suitable amino acid residues that are responsible for catalysis of the reaction. Cofactors that are also involved in the reaction are usually bound at or near the active site.
4. Enzyme-substrate [ES] complex: Enzymatic reactions proceed through formation of a transition state i.e. an intermediate state between substrate and product having higher free energy than that of either the substrate or product. This state results from binding of the substrate to the active site of the enzyme resulting in an [ES] complex.
1. Fischer’s Lock-and-Key hypothesis: Emil Fischer in 1894 postulated that enzymes and their substrates possess specific complementary geometric shapes that fit exactly into each other like a key fits in a lock. This model accounts for the specificity of enzymes but fails to account for stabilization of the transition state.
Enzymes: Basic Concepts and Kinetics
1. Michaelis-Menten enzyme kinetics: A simple model to explain the kinetic characteristics of enzymatic reactions was proposed by Leonor Michaelis and Maud Menten in 1913. According to this model, formation of the [ES] complex intermediate is essential for the enzymatic catalysis reaction to take place. A group of enzymes that does not obey the Michaelis-Menten enzyme kinetics is comprised of the allosteric enzymes. These enzymes have multiple active sites and binding of substrate to one site affects, positively or negatively, the binding of substrate to the remaining sites.
2. Rate constants: An enzyme [E] combines with its substrate [S] to form the enzyme-substrate complex [ES] with a rate constant of k1. This complex can either form the product [P] with rate constant k2 or can dissociate to undergo a reverse reaction with rate constant
Kinetics of enzymatic reactions
k-1 which results in release of the substrate.1 In the Michaelis-Menten model for enzyme kinetics, it has been assumed that dissociation of the [ES] complex to give back the substrate is negligible. This condition applies for the initial stages of a reaction when the equilibrium favours product formation
3. Reaction velocity (Vo): The rate of increase in product concentration [P] with time when the concentration is low is known as the reaction velocity (Vo). It is also often defined as the number of moles of product formed per second. Vo is linearly proportional to substrate concentration [S] when [S] is low but becomes independent of [S] when [S] is high.
Enzymes: Basic Concepts and KineticsMolecular & cell Biology
4. Vmax: The maximal rate that can be achieved by an enzyme when all its catalytic sites are occupied by the substrate is referred to as Vmax or maximum velocity.
5. Km: The substrate concentration at which the reaction rate is half its maximal value is known as Km or the Michaelis constant. Km value of an enzyme is an indicator of the affinity that the enzyme has for its substrate or in other words, it is a measure of the strength of the [ES] complex. A high Km indicates weak affinity between enzyme and substrate while a low Km is indicative of strong affinity. Michaelis constant. Km value of an enzyme is an indicator of the affinity that the enzyme has for its substrate or in other words, it is a measure of the strength of the [ES] complex. A high Km indicates weak affinity between enzyme and substrate while a low Km is indicative of strong affinity.
Kinetics of enzymatic reactions
6. Enzyme activity: The activity of an enzyme can be defined either in International Units (U) or Katal. 1 U of enzyme activity is defined as the amount of enzyme that catalyzes the conversion of 1 mmole of substrate into product at 25oC under the specified assay conditions. 1 Katal is the amount of enzyme that catalyzes the conversion of 1 mole of substrate into product per second. (1 U = 16.67 nanokatal)
7. Specific activity: This can be defined as the enzyme activity per milligram of protein. It is a measure of enzyme purity and increases continuously during a purification process until it achieves a maximum, constant value indicating the pure enzyme state.
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
8. Turnover number (kcat): The turnover number of an enzyme can be defined as the maximum number of substrate molecules that can be converted into product per active site of the enzyme per unit time. Unit of kcat is s-1 or min-1 and can obtained from parameters of the Michaelis-Menten equation (Vmax/[E]).
Kinetics of enzymatic reactions
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
1. Enzyme inhibition: Several molecules are capable of binding at or near the active site of enzymes thereby decreasing or inhibiting their activity. It provides an important control mechanism in biological systems. Enzyme inhibition is also an important mechanism that is exploited during the manufacture of various drug molecules.
2. Reversible inhibition: Inhibition of an enzyme can be reversed if there is rapid dissociation of the enzyme-inhibitor complex. The inhibitor is associated to the enzyme molecule by relatively weaker interactions.
3. Irreversible inhibition: In case of irreversible inhibition, the inhibitor covalently modifies the enzyme and dissociates very slowly from the target enzyme because it is tightly bound. The action of penicillin,
Enzyme inhibition
an important antibiotic, is through the irreversible inhibition of the enzyme transpeptidase that is essential for bacterial cell wall synthesis. Aspirin, the commonly used analgesic and anti-pyretic also functions by means of irreversible inhibition of the enzyme cyclooxygenase.
4. Competitive inhibition: This is a type of reversible inhibition wherein the inhibitor binds to the active site of the enzyme thereby preventing the substrate from binding to it. The inhibitor and substrate, in this case, are structural analogues and the reaction rate is decreased due to fewer enzyme molecules being bound to the substrate. This type of inhibition can be overcome by increasing the substrate concentration.
Molecular & cell BiologyEnzymes: Basic Concepts and Kinetics
5. Uncompetitive inhibition: In this case, the substrate and the inhibitor have distinct binding sites on the enzyme and the inhibitor binds only to the ES complex and not to the enzyme alone. Both Km and Vmax are found to decrease in this type of inhibition.
6. Mixed inhibition: A mixed inhibitor also binds to the enzyme at a site distinct from the substrate binding site with the difference being that it can bind either to E or ES. Binding of either one brings about conformational changes in the enzyme structure thereby affecting binding of the other.This inhibition can be reduced but not overcome by increasing substrate concentration. Non-competitive inhibition is a special case of mixed inhibition where inhibitor binding reduces catalytic activity but does not affect substrate binding.
