Enzymes Activity MGD S3 Lecture 1 PDF

Enzymes Activity Effect of Enzyme on Chemical Reaction •There are two fundamental conditions for life. First, the living thing must be able to self- replicate. Second, the organism must be able to catalyze chemical reactions efficiently and selectively. > •Enzymes are proteins that act as catalysts, c...

Enzymes Activity Effect of Enzyme on Chemical Reaction •There are two fundamental conditions for life. First, the living thing must be able to self- replicate. Second, the organism must be able to catalyze chemical reactions efficiently and selectively. > •Enzymes are proteins that act as catalysts, compounds that increase the rate of chemical reactions. • Enzyme catalysts bind reactants (substrates), convert them to products, and release the products. •Although enzymes may be modified during their participation in this reaction sequence, they return to their original form at the end. 2. Enzymes are highly specific Interact with one or only a few substrates and catalyse one type of reaction. Enzymes classification: Enzymes can be grouped into one of six main classes: 3. Enzymes increase the rate of a reaction Enzymes increase the rate of the reaction by factors of 1 million or more. They DO NOT affect the equilibrium of a reaction. 1. Oxidoreductases: Oxidation-reduction reactions (i.e. transfer of electrons). 2. Transferases: Transfer C-, N- or P- containing groups 4. Enzymes are left unchanged after the reaction has occurred 3. Hydrolases: Catalyse cleavage of bonds by the addition of water Note: Enzymes provide a place for the reaction occur. Although there may be changes to the enzyme during the course of the reaction on completion the enzyme will be unchanged. 4. Lyases: Addition or removal of groups to form double bonds - • Isoenzymes: enzymes that catalyze the same reaction but have a different amino acid sequence. 5. Isomerases: Transfer of groups within molecules to form isomers How do Enzymes Work? 6. Ligases: Formation of bonds between C and O, S or N at the expense of ATP •Enzyme-catalyzed reactions are characterized by the formation of a complex between substrate and enzyme (an ES complex). Enzyme Nomenclature Enzymes are named by the type of reaction that they catalyse. Usually this means adding the suffix –ase to the name of their substrate or reaction that they catalyse. •Substrate binding occurs in a pocket on the enzyme called the active site. e.g/ • Lactase hydrolyses lactose into glucose and galactose " - •DNA polymerase, polymerises deoxynucleotides to form DNA Properties of enzymes: 1. Virtually all enzymes are proteins Some enzymes also require the presence of additional chemical components to catalyse reactions. - • Cofactors are inorganic ions such as Fe2+, Mn2= etc. • Coenzymes are organic compounds that act as temporary carriers of groups in the reaction e.g. nicotinamide adenine dinucletide (NAD), Coenzyme A (CoA). •Coenzymes or cofactors that are tightly or covalently linked to the enzyme protein are known as prosthetic groups. •Enzymes work by lowering the activation energy needed for a reaction to occur. Binding of substrate to a distinct part of the enzyme, the active site, increases the local concentration of reactants and also stabilizes the formation of the high energy transition state. The active site •The active site of an enzyme is the place where the reaction occurs. •Although enzymes usually contain at least 100 amino acids the active site is composed of only a few of these. The rest of the molecule acts as a scaffold to allow the correct positioning of the amino acids making up the active site. The Michaelis-Menten Model for enzyme catalysis Effect of substrate concentration: An enzyme, E, combines reversibly with substrate, S, to form an enzyme-substrate intermediate, ES. ES can then break down to form free enzyme and the reaction product P. •The rate or velocity of a reaction (v) is the number of substrate molecules converted to product per unit time; velocity is usually expressed as μmol of product formed per minute. - •The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached. (Increase in the rate of reaction to a certain point) - - •Active sites are usually clefts or crevices in the protein. This allows substrates to bind and for water to be excluded from the reaction. Binding of the substrate to the active site is an important determinant of specificity. Only molecules that have a complementary shape to the active site will be able to bind (Lock and Key Hypothesis). •The substrate is held in the active site by multiple weak bonds with amino acids in this part of the enzyme. Often binding of a substrate results in changes in the shape of the enzyme to enhance binding (Induced Fit Hypothesis). From this we can derive the Michaelis-Menten equation which describes how the reaction velocity V0 varies with the substrate concentration. Where : V0 = initial reaction velocity [S] = substrate concentration Vmax = maximal velocity Km = Michaelis constant Km: the substrate concentration that will give half the maximal rate (Vmax). •Low Km means high affinity of the enzyme to the substrate. •Most enzymes show Michaelis-Menten kinetics, in which the plot of initial reaction velocity (vo) against substrate concentration ([S]), is hyperbolic (similar in shape to that of the oxygen-dissociation curve of myoglobin). •In contrast, allosteric enzymes do not follow Michaelis-Menton kinetics and show a sigmoidal curve that is similar in shape to the oxygen dissociation curve of hemoglobin. •High Km means low affinity of the enzyme to the substrate. Effect of enzyme concentration: Lineweaver-Burk Equation •The Michaelis-Menten equation can be rearranged to give a linear plot: The rate of a reaction is directly proportional to the concentration of enzyme; if you double the amount of enzyme, you will double the amount of product produced per minute, whether you are at low or at saturating concentrations of substrate. Effect of Temperature : w •Allows for easy estimation of Km and Vmax from linear plot. The reaction velocity increases with temperature until a peak velocity is reached. Further elevation of the temperature results in a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme. • The optimum temperature for most human enzymes is between 35 and 40°C. Human enzymes start to denature at temperatures above 40°C. - . - Inhibition of enzyme activity Effect of pH The catalytic process usually requires that the enzyme and substrate have specific chemical groups in either an ionized or unionized state in order to interact. •For example, catalytic activity may require that an amino group of the enzyme be in the protonated form (NH3+). At alkaline pH, this group is deprotonated, and the rate of the reaction, therefore, declines. Also extremes of pH can also lead to denaturation of the enzyme. We have two types of enzyme inhibition : •Irreversible inhibition •Reversible inhibition •Knowing how enzyme inhibition occur is very important as Many drugs work by inhibiting the activity of enzymes. 1. Irreversible inhibitors Bind covalently to the enzyme molecule to destroy enzyme function •An irreversible inhibitor inactivates an enzyme by bonding covalently to a particular group at the active site. The inhibitor-enzyme bond is so strong that the inhibition cannot be reversed by the addition of excess substrate 2. Reversible inhibitors Typically bind to enzymes through noncovalent bonds. The two most commonly encountered types of reversible inhibition are competitive and noncompetitive. Non-competitive inhibitors: •A type of enzyme inhibition where the inhibitor binds at a site other than the active site. •Noncompetitive inhibition, a type of allosteric regulation, is a specific type of enzyme inhibition characterized by an inhibitor binding to an allosteric site resulting in decreased efficacy of the enzyme. • An allosteric site is simply a site that differs from the active site-where the substrate binds This type of inhibition: •Affects Vmax not Km •Cannot be overcome by increasing the substrate concentration. ⇐ E- Competitive inhibitors: International unit of Enzyme Activity •In some diseases, there may be a deficiency or even a total absence of one or more enzymes. For other disease conditions, excessive activity of an enzyme may be the cause. •Measurements of the activities of enzymes in blood plasma, erythrocytes, or tissue samples are important in diagnosing certain illnesses. •Enzyme Activity: the amount of product formed by per unit Activity = amount of product / time •By international agreement, 1.0 unit of enzyme activity is defined as the amount of enzyme causing transformation of 1.0 micromol of substrate per minute or the amount of enzyme that produces 1 μmol of product per minute. under optimal conditions of measurement. A type of enzyme inhibition where the inhibitor which bears a structural resemblance to a particular substrate competes with the substrate for binding at the active site. So that it prevent the substrate from approaching the active site This type of inhibition: •Affects Km not Vmax • Can be overcome by increasing the substrate concentration Examples of competitive inhibitors: • Allopurinol competes with hypoxanthine for xanthine oxidase inhibiting the formation of uric acid, so it is used in treatment of hyperuricemia (gout). • Statins (e.g. atorvastatin) competes with HMGCoA for its reductase, so, it inhibits cholesterol synthesis. 1 unit of activity = 1 μmol/min Hemi1n " O