Lecture 9: Enzymes PDF

LECTURE - 9 ENZYMES Biochemistry Lecture by Dr. Siti Norhawani Harun, Lecturer, Faculty of Pharmacy, MAHSA University , 0  Lecture outline Properties of enzyme Classification of enzyme Cofactors, Co-substrate, Co-enzymes...

LECTURE - 9 ENZYMES Biochemistry Lecture by Dr. Siti Norhawani Harun, Lecturer, Faculty of Pharmacy, MAHSA University , 0  Lecture outline Properties of enzyme Classification of enzyme Cofactors, Co-substrate, Co-enzymes Enzyme mechanism Active site of enzyme Factor affecting reaction velocity Michaelis-Menten equation & Lineweaver-Burk plot Enzyme inhibitors  Properties of Enzyme Specialized protein that catalyzed biological reactions The active site of enzyme contains amino acid side chains that create a 3-dimensional surface complementary to the substrate. Enzyme + substrate →enzyme-substrate complex (ES) → enzyme-product complex (EP) →enzyme + product Enzyme-catalyzed reactions are highly efficient - each enzyme molecule is capable of transforming 100 – 1000 substrate molecules into product each second. Turnover number, Kcat = number of molecules of substrate converted to product per enzyme molecule per second.  International Union of Biochemistry and Molecular Biology (IUBMB) : 6 classes of enzyme according to the chemical reactions catalysed.  1. Oxidoreductase Catalyze oxidation-reduction reactions Oxidation – loss of electron Reduction – addition of electron catalyzes the transfer of electrons from one molecule (the reductant, also called the hydrogen or electron donar) to another (the oxidant, also called the hydrogen or electron acceptor) usually utilizes NADP or NAD as cofactors.  2. Transferases transfer a chemical group (containing C-, N- or P-) from one molecule to another substrates and products A–X + B →A + B–X A would be the donor, and B would be the acceptor. The donor is often a coenzyme  coenzymes - can be tightly bound or can be covalently attached to the enzyme. - Some are modified during a reaction but are in their original state at the end of the reaction. - Some are modified at the end, but they must participate in another reaction to be returned to their original state.  Enzyme mechanism 1. Binding of Substrate by an Enzyme Two theories I) lock-and key-theory II) Induced fit theory  Active site of enzyme The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that participate in recognition of the substrate. Substrates bind to the active site of the enzyme or a specificity pocket through hydrogen bonds, hydrophobic interactions, temporary covalent interactions (van derWaals) or a combinationof all of these to form the enzyme-substrate complex.  Residues of the active site will act as donors or acceptors of protons. or other groups on the substrate to facilitate the reaction. In other words, the active site modifies the reaction mechanism in order to change the activation energy of the reaction. The product is usually unstable in the active site and will be released and return the enzyme to its initial unbound state.  2. Transition state In order for a reaction to occur, reactant molecules must contain sufficient energy to cross a potential energy barrier, the free energy of activation. In the absence of an enzyme, only a small proportion of a population of molecules may possess enough energy to achieve the transition state between reactant and product. The lower the free energy of activation, the more reactants have sufficient energy to pass through the transition state, and, hence, the faster the rate of the reaction. The transition state is where the bond is starting to form and the geometry is changing.  Factor The rate orAffecting Reaction velocity of a reaction Velocity (V) is the number of substrate molecules converted to product per unit time. Velocity usually expressed as µmol of product formed per minute. 1. Substrate concentration The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) The leveling off of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present.  2. Temperature Increase of velocity with temperature increases number of molecules having sufficient energy to pass over the energy barrier and form the products of reaction. Further elevation of temperature will denature the enzyme and leads to decrease in reaction velocity Temperature optimum for mammalian enzyme: 40 – 45 °C  In order to model an enzymatic reaction, some conditions must be maintained. Temperature, ionic strength, pH, and other physical conditions that might affect the rate must remain constant. Each enzyme can act on only one other molecule at a time. The enzyme must remain unchanged during the course of the reaction.  The concentration of substrate must be much higher than the concentration of enzyme [ES] does not change with time (steady-state assumption). The rate of [ES] formation is equal to that of the breakdown of ES (to E+S and to E+P) The rate of reaction is measured as soon as enzyme & substrate are mixed.  Meaning of Km (Michaelis constant) Km is numerically equal to the [S] at which the reaction velocity is equal to ½Vmax. Does not vary with the concentration of enzyme, used to indicate how well a substrate interacts with an enzyme (reflects the affinity of the enzyme for that substrate). The smaller the value of Km, the tighter is the interaction between substrate and enzyme. E.g. A Km of 10-7M indicates that the substrate has a great affinity for the enzyme than if the Km is 10-5M  Turnover number (Kcat) Thelarger The number the of molecules value ofof Kcat substrate forconverted to product an enzyme, the per unit time per molecule of enzyme. faster the reaction will be. Relationship of velocity to enzyme concentration Rate of reaction α enzyme concentration. E.g. if the [E] is halved, the initial rate of reaction (Vo), as well as Vmax, are reduced to half that of the original. Small Km : high affinity, a low [S] is needed to half-saturated the enzyme. Large Km: low affinity, high [S] is needed to half-saturated the enzyme  Enzyme Inhibitors Inhibitors – any substance that can diminish the velocity of an enzyme-catalyzed reaction 1. Irreversible Very tightly bound to enzyme, either covalently or noncovalently, but effectively don't come off Chemically modified & inactivate an enzyme Irreversible inhibitors are usually considered to be poisons and are generally unsuitable for therapeutic purposes.  2. Reversible rapid binding/noncovalent bonds, release from enzyme in an equilibrium reversible inhibitors are divided into 3 categories: competitive inhibitors, noncompetitive inhibitors & uncompetitive inhibitor.  2) Penicillin (an antibiotic) both a transition-state analog and a suicide substrate covalently inhibits a transpeptidase which involved in bacterial cell wall synthesis (eukaryotic cells don't have this enzyme). Normal transpeptidase catalytic mechanism: substrate bound to the Ser-OH of enzyme, making a covalent acyl-enzyme intermediate -- Covalent intermediate continues in enzyme-catalyzed reaction to form peptide cross-link in peptidoglycan structure of cell wall, regenerating free enzyme for another round of catalysis. Penicillin resembles transition state in structure, so penicillin binds very tightly and very reactive. THANK YOU