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ENZYMES

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  1. ENZYMES Medical Biochemistry, Lecture 23

  2. Lecture 23, Outline • Definition of enzyme terms and nomenclature • Description of general properties of enzymes • Binding energy and transition states • Catalytic mechanisms and functional groups • In book, Chapters 8,10; Ignore pp 78-80 • Recommended supplement for lectures 23-25: UNDERSTAND Biochemistry CD

  3. Enzyme Catalysis Overview

  4. Enzyme Nomenclature • active site- a region of an enzyme comprised of different amino acids where catalysis occurs (determined by the tertiary and quaternary structure of each enzyme) • substrate - the molecule being utilized and/or modified by a particular enzyme at its active site • co-factor - organic or inorganic molecules that are required by some enzymes for activity. These include Mg2+, Fe2+, Zn2+ and larger molecules termed co-enzymes like nicotinamide adenine dinucleotide (NAD+), coenzyme A, and many vitamins.

  5. Enzyme Nomenclature (cont) • prosthetic group - a metal or other co-enzyme covalently bound to an enzyme • holoenzyme - a complete, catalytically active enzyme including all co-factors • apoenzyme - the protein portion of a holoenzyme minus the co-factors • isozyme - (or iso-enzyme) an enzyme that performs the same or similar function of another enzyme. This generally arises due to similar but different genes encoding these enzymes and frequently is tissue-type specific or dependent on the growth or developmental status of an organism.

  6. Clinical Use of Enzymes • Enzyme Activity in Body Fluids Reflects Organ Status: • Cells die and release intracellular contents; increased serum activity of an enzyme can be correlated with quantity or severity of damaged tissues (ex. creatine kinase levels following heart attack) • Increased enzyme synthesis can be induced and release in serum correlates with degree of stimulation (ex. alkaline phosphatase activity as a liver status marker)

  7. Clinical Use of Enzymes (cont) • Enzyme Activity Reflects the Presence of Inhibitors or Activators • Activity of serum enzymes decreases in presence of an inhibitor (ex. some insecticides inhibit serum cholinesterases) • Determine co-factor deficiencies (like an essential vitamin) by enzyme activity (ex. add back vitamin to assay, if activity increases, suggests deficiency in that vitamin)

  8. Clinical Use of Enzymes (cont) • Enzyme activity can be altered genetically • A mutation in an enzyme can alter its substrate affinity, co-factor binding stability etc. which can be used as a diagnostic in comparison with normal enzyme • Loss of enzyme presence due to genetic mutation as detected by increased enzyme substrate and/or lack of product leading to a dysfunction • NOTE: PCR techniques that identify specific messenger RNA or DNA sequences are replacing many traditional enzymatic based markers of genetic disease

  9. ENZYMATIC REACTION PRINCIPLES • Biochemically, enzymes are highly specific for their substrates and generally catalyze only one type of reaction at rates thousands and millions times higher than non-enzymatic reactions. Two main principles to remember about enzymes are 1) they act as CATALYSTS (they are not consumed in a reaction and are regenerated to their starting state) and 2)they INCREASE THE RATE of a reaction towards equilibrium (ratio of substrate to product), but they do not determine the overall equilibrium of a reaction.

  10. CATALYSTS • A catalyst is unaltered during the course of a reaction and functions in both the forward and reverse directions. In a chemical reaction, a catalyst increases the rate at which the reaction reaches equilibrium, though it does not change the equilibrium ratio. For a reaction to proceed from starting material to product, the chemical transformations of bond-making and bond-breaking require a minimal threshold amount of energy, termed activation energy. Generally, a catalyst serves to lower the activation energy of a particular reaction.

  11. ENZYMATIC REACTION PRINCIPLES (cont) • The energy maxima at which the reaction has the potential for going in either direction is termed the transition state. In enzyme catalyzed reactions, the same chemical principles of activation energy and the free energy changes (DGo) associated with catalysts can be applied. Recall that an overall negative DGo indicates a favorable reaction equilibrium for product formation. As shown in an enzyme catalyzed reaction, and in the graph, the net effect of the enzyme is to lower the activation energy required for product formation.

  12. Chemical Reaction

  13. Enzymatic Reaction Energetics

  14. Reaction Rates • The rate of the reaction is determined by several factors including the concentration of substrate, temperature and pH. For most standard physiological enzymatic reactions, pH and temperature are in a defined environment (pH 6.9-7.4, 37oC). Therefore, the concentration of substrate is the critical determinant. This enzymatic rate relationship has been described mathematically by combining the equilibrium constant (the ratio of substrate and product concentrations), the free energy change and first or second-order rate theory. The net result for enzymatic reactions is that the lower the activation energy, the faster the reaction rate, and vice versa.

  15. Binding Energy • The graph of activation energy and free energy changes for an enzymatic reaction also indicates the role binding energy plays in the overall process. Due to the high specificity most enzymes have for a particular substrate, the binding of the substrate to the enzyme through weak, non-covalent interactions is energetically favorable and is termed binding energy. The same forces important in stabilizing protein conformation (hydrogen bonding and hydrophobic, ionic and van der Waals interactions) are also involved in the stable binding of a substrate to an enzyme.

  16. Binding Energy and Transition State • The cumulative binding energies from the non-covalent interactions are optimized in the transition state and is the major source of free energy used by enzymes to lower activation energies of reactions. A single weak interaction has been estimated to yield 4-30 kJ/mol energy, thus multiple interactions (which generally would occur during binding and catalysis) can yield up to 60-80 kJ/mol free energy - this accounts for the large decreases in activation energies and increases in rate of product formation observed in enzymatic-catalyzed reactions.

  17. Effect of Temperature A reaction rate will generally increase with increasing Temperature due to increased kinetic energy in the system until a maximal velocity is reached. Above this maximum, the kinetic energy of the system exceeds the energy barrier for breaking weak H-bonds and hydrophobic interactions, thus leading to unfolding and denaturation of the enzyme and a decrease in reaction rate.

  18. Effect of pH Variations in pH can affect a particular enzyme in many ways, especially if ionizable amino acid side chains are involved in binding of the substrate and/or catalysis. Extremes of pH can also lead to denaturation of an enzyme if the ionization state of amino acid(s) critical to correct folding are altered. The effects of pH and temperature will vary for different enzymes and must be determined experimentally.

  19. LOCK-AND- KEY INDUCED FIT

  20. Hexokinase Active Site: Glucose vs. Galactose Binding

  21. Co-factor: NAD+/NADH (EXAMPLE)

  22. Co-factors: Co-A and Biotin

  23. Catalytic Mechanisms: Types • Four types of catalytic mechanisms will be discussed: • binding energy catalysis • general acid-base catalysis • covalent catalysis • metal ion catalysis

  24. Acid-Base Catalysis Many reactions involve the formation of normally unstable, charged intermediates. These intermediates can be transiently stabilized in an enzyme active site by interaction of amino acid residues acting as weak acids (proton donors) or weak bases (proton acceptors). The general acid and general base forms of the most common and best characterized amino acids involved in these reactions are shown above.

  25. Acid-Base Catalysis (cont) • The preceding functional groups can potentially serve as either proton donors or proton acceptors. This is dependent on many factors including the molecular nature of the substrate, any co-factors involved, and the pH of the active site (which would determine the ionization state of an amino acid side chain). For acid-base catalysis, histidine is the most versatile amino acid due to its pKa which means that in most physiological situations it can act as either a proton donor or proton acceptor. Generally these amino acids will interact together with the substrate, or in conjunction with water or other weak, organic acids and bases found in cells.

  26. Binding Energy Catalysis • Binding energy accounts for the overall lowering of activation energy for a reaction, and it can also be considered as a catalytic mechanism for a reaction. Several catalytic factors in the binding of a substrate and enzyme can be considered: 1) transient limiting of substrate and enzyme movement by reducing the relative motion (or entropy) of the two molecules, 2) solvation disruption of the water shell is thermodynamically favorable, and 3) substrate and enzyme conformational changes. All three of these factors individually or in combination are utilized to some degree by an enzyme. While in some instances these forces alone can account for catalysis, they are frequently components of a complex catalytic process involving factors discussed for the other types of catalytic mechanisms.

  27. Covalent Catalysis • This mechanism involves the transient covalent binding of the substrate to an amino acid residue in the active site. Generally this is to the hydroxyl group of a serine, although the side chains of threonine, cysteine, histidine, arginine and lysine can also be involved.

  28. Metal Ion Catalysis • Various metals, all positively charged and including zinc, iron, magnesium, manganese and copper, are known to form complexes with different enzymes or substrates. This metal-substrate-enzyme complex can aid in the orientation of the substrate in the active site, and metals are known to mediate oxidation-reduction reactions by reversible changes in their oxidation states (like Fe3+ to Fe2+).

  29. Summary of Catalytic Mechanisms • In general, more than one type of catalytic mechanism will occur for a particular enzyme via various combinations of binding energy, acid-base, covalent and metal catalysis. Enzymes as a whole are incredibly diverse in their structures and the types of reactions that they catalyze, therefore there is also a large diversity of catalytic mechanisms utilized, the basis of which must be determined experimentally.

  30. Substrate Binding Pockets of Chymotrypsin and Trypsin

  31. Catalytic Mechanism of Chymotrypsin

  32. Chymotrypsin Mechanism (cont)

  33. Chymotrypsin, last step and regeneration of active enzyme