Enzymes PDF

Welcome Enzymes By Prof. Dr Nesreen Elhelbawy Medical Biochemistry and Molecular Biology Department Faculty of Medicine, Menoufia University Intended learning outcomes (ILOs): By the end of the chapter, students should be able to: 1-Describe the classification, n...

Welcome Enzymes By Prof. Dr Nesreen Elhelbawy Medical Biochemistry and Molecular Biology Department Faculty of Medicine, Menoufia University Intended learning outcomes (ILOs): By the end of the chapter, students should be able to: 1-Describe the classification, nomenclature and structure of enzymes. 2-Identify coenzymes, isoenzymes and cofactors. 3-Understand mechanism of action and factors affecting the rate of enzyme action Enzymes: Biological catalysts. They accelerate reactions and undergo changes during the reaction but revert to their original state when the reaction is complete. Enzymes increase the reaction rate by lowering the free energy barrier that separates the reactants and products.  Mechanism of action of enzymes Catalysis acts by decreasing the energy barrier between reactants and products, making it easy to reach the transition state. The transition state is the transitory of molecular structure in which the molecule is no longer a substrate but not yet a product  Enzyme specificity 1-Absolute specificity: Enzymes catalyze only one specific reaction on one molecule e.g. uricase, urease, catalase specifi c a 2-Relative Specificity: One enzyme acts on group of compound having the same type of bonds e.g lipase acts on ester bond of triacylglycerols and phospholipids.  Enzyme classification The six classes of enzymes include: 1- Oxidoreductases: Enzymes catalyzing oxidation – reduction reactions ( e.g. alcohol dehydrogenase) + + Alcohol + NAD Aldehyde or ketone + NADH+ H 2- Transferases: Enzymes catalyzing transfer of functional groups (other than H) between two substrates (choline acyltransferase). Acetyl- CoA + Choline CoA + Acetylcholine 3- Hydrolases: Enzymes catalyzing hydrolysis of a bond by addition of water (peptidases and proteinases e.g. trypsin and chymotrypsin). 4- Lyases: Enzymes catalyzing group elimination to form double bonds (fumarase). L-malate fumarate + H2O 5- Isomerases: Enzymes catalyzing interconversion of isomers by rearrangement of atomic groupings without altering molecular weight or number of atoms (aldoses and ketoses). 6- Ligases: Enzymes catalyzing bond formation coupled with ATP hydrolysis. ATP + Acetyl CoA + CO2 ADP + Pi + Malonyl- CoA. (acetyl-CoA carboxylase)  Enzyme nomenclature classification of enzymes according to the chemical reaction type and reaction mechanisms. Each enzyme has a code number (EC) that classify the reaction type to class (first digit), subclass (second digit) and subsubclass (third digit). The fourth digit is for the specific enzyme. EC 2. 7. 1. 1. Class 2: a transferase. Subclass 7: transfer phosphate. Subsubclass 1: the phosphate acceptor is alcohol. Final digit: denotes the enzyme hexokinase.  Cofactors and coenzymes Some enzymes needs small molecule cofactors to do its action. These cofactors includes: 2+ 3+ 2+ 1) Metal ions (Cu , Fe or Zn ). 2) Organic molecules (coenzymes) such as : Cosubstrate (transiently associated with enzyme). Prothetic groups (permanently associated with enzyme. e.g. heme prothetic group of cytochrome c is tightly bound to the protein). Apoenzyme (inactive) + cofactor ! holoenzyme (active).  Types of coenzymes Coenzymes can be classified to (according to group transferred): 1- Coenzymes for transfer groups other than hydrogen: Pyridoxal phosphate (B6) for amino group Tetrahydrofolate for one carbon group transfer. Thiamin pyrophosphate (B1) for aldehyde transfer. Biocytin (from biotin) for carboxylation reactions. 2- Coenzymes for transfer of hydrogen: + Nicotinamide coenzymes ( ) from niacin for + oxidation-reduction reactions. NAD and NADP Flavin coenzymes (FMN and FAD) from riboflavin (B2) for oxidation-reduction reactions. Coenzyme Q.  APOENZYME and HOLOENZYME Conjugated protein enzyme (holoenzyme): it consists of Protein part of the enzyme known as apoenzyme. Non protein part known as cofactor.  Active site of enzymes The catalytic (active) site: Is a restricted region of the enzyme at which catalysis takes place. It is a pocket or groove in the surface of the protein (which attract substrate and mediate catalysis). There are 2 models of the catalytic site.  Catalytic site models: 1- Lock & key or rigid template model Substrate and enzyme interact like lock and key. The substrate binds to a specific site on the enzyme. The shape of that site is complementary to that of the substrate.  2- Induced fit model The substrate induces a conformational change in the enzyme, making the enzyme in the correct orientation for substrate binding, catalysis or both.  Isoenzymes Definition: These are physically distinct forms of a given enzyme which are present in different cell types (different tissues). They have multiple form but has the same catalytic activity. Differ in their electrophoresis mobility Isoenzymes may differ in their affinity for substrates. Examples of isoenzymes: Lactate dehydrogenase enzyme (LDH), which can be distinguished on the basis of their electrophoretic mobilities into 5 isoenzymes (LDH1- 5).  LDH isoenzymes Each isoenzyme is a tetramer (fromed from two types of polypeptide, H and M). H and M subunits are expressed in different tissues. LDH 1 (HHHH), LDH2 (HHHM), LDH3 (HHMM), LDH4 (HMMM) and LDH5 (MMMM). LDH1 and LDH2 are predominant in the heart and red cells (increased in MI), whereas LDH4 and LDH5 are predominant in the liver and some skeletal muscles.  Factors affecting enzyme activity 1 Temperature. 2 PH. 3 Enzyme concentration. 4 Substrate concentration. 5 Inhibitors. (1) Temperature: Over a limited range of T, the velocity of an enzyme-catalyzed reaction is increased as the T raises. Optimal temperature: It is the T at or above that of the cells in which the enzymes occur. At this T the reaction is rapid, above it the reaction rate decreases (enzyme denaturation) (2) pH: Moderate pH changes affect the ionic state of the enzyme and substrate. The optimal enzyme activity ranges between 5-9, except few enzymes (e.g. pepsin). (3) Enzyme concentration: The velocity of an enzyme-catalyzed reaction is directly proportional to the enzyme concentration. The rate of the reaction is initially constant but gradually decreases (depletion of substrate, inhibition of the enzyme by its product or enzyme denaturation). So, only the initial velocity (Vi) is used to calculate the kinetic parameters of the reaction. Enzymes change the reaction path but do not affect the concentrations of the reactants and products. (4) Substrate concentration: - The velocity of an enzyme-catalyzed reaction is increased with increased substrate concentration up to a point where the enzyme becomes saturated with the substrate. V - Vi (initial velocity) increases and reach max (maximum velocity) which is not affected by further increase in S concentration. K1 K2 Enz + S EnzS Enz + P ∀K -1 - Mechaelis-Menten equation describe the relation between Vi, S, Vmax and rate constants K1, K-1 and K2. Vmax [S] Vi = [S] + K-1 + K2 K1 - Km is the substrate concentration that produces half maximal velocity (Vmax /2). Km = K-1 + K2 K1 Vmax [S] Vi = [S] + Km Significance of Mechaelis-Menten constant : When [S] = Km Vmax [S] Vi = [S] + [S] V Vi = max 2 * The Km is unique for each enzyme-substrate pair. Lineweaver-Burk or double reciprocal plot: - A good method for determining the values of Vmax and Km is the reciprocal of the Mechaelis-Menten equation. Vmax [S] Vi = [S] + Km 1 [S] +V Km Vi = max [S] - This is the equation of a straight line. When these quantities are plotted, we will obtain the Lineweaver-Burk or double reciprocal plot.  (5) Inhibitors Definition:These are substances that reduce enzyme’s activity, it combines with the enzyme either reversibly or irreversibly. (1) Irreversible inhibitiors irreversible inhibitors are those : that bind covalently with enzyme or destroy a functional group on an enzyme => that is essential for enzyme’s activity. (2) Reversible inhibitors: Competitive Uncompetitive Non-competitive A- Competitive inhibitors It is a substance that competes directly with a substrate for an enzyme’s substrate-binding site. The chemical structure of the inhibitor (I) resembles that of the substrate (S). High concentration of the S can overcome the effects of the I. It increases the Km of the enzyme but has no effect on the Vmax (In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that were reached in its absence).  Regulation of enzyme activity (A) Control of enzyme availability: The amount of a given enzyme in a cell depends on: The rate of its synthesis The rate of its degradation # 1- Regulation of enzyme synthesis Regulation of enzyme synthesis Enzyme Enzyme repression induction Product Catabolite Inducers repression repression  2- Regulation of enzyme degradation Increase enzyme amout by decrease rate of degradation or decrease in amount of enzyme by increase the rate of degradation - Example Liver arginase level is increased in: - Starvation in animal due to decreased arginase degradation. B-Control of enzyme activity 1Allosteric effectors 2Covalent modification 3Limited proteolysis (B) Control of enzyme activity: 1- Allosteric effectors: - Enzymes bind the effector at an allosteric site that is physically distinct from catalytic site. Allosteric enzymes: are enzymes whose activity at the catalytic site may be modulated by the presence of allosteric effectors at the allosteric site. Feedback inhibition Inhibition of the activity of an enzyme in a biosynthetic pathway by an end product of that pathway. 123 A B C D High concentration of D inhibits conversion of A to B. So, D acts as a negative allosteric effector or feedback inhibitor of the enzyme, regulating the synthesis of D. 2- Covalent modification: The catalytic activity of an enzyme can be modulated by covalent attachment of a phosphate group These enzymes are termed interconvertable enzymes. The phospho- or dephosphoenzyme may be the more active catalyst. Protein kinases catalyze phosphorylation and protein phosphatases catalyze dephosphorylation (hormonal and neural control).  3- Limited proteolysis: Enzyme activity can be regulated by converting an inactive proenzyme to a catalytically active form. The proenzyme must undergo limited proteolysis, which is associated with conformational changes that create the catalytic site. Examples of proenzymes are: Digestive enzymes. Blood coagulation enzymes. Blood clotting enzymes.