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  2. 2. Molecular Interactions In Cell Events <ul><li>CATALYSIS : </li></ul><ul><li>- The vast number of coordinated and complex biochemical reactions that occur in an organism is summarised as the cell METABOLISM </li></ul><ul><li>- The reactions are in ordered pathways, controlled at each stage by ENZYMES </li></ul><ul><li>- Through these metabolic pathways, the cells are able to transform energy, breakdown macromolecules and synthesise new organic molecules needed for life </li></ul>
  3. 3. Anabolic Reactions <ul><li>Uses energy to SYNTHESISE large molecules from smaller ones e.g. </li></ul><ul><li>Amino Acids->Proteins </li></ul><ul><li>Also known as endothermic reactions </li></ul>ENDOTHERMIC REACTION
  4. 4. Catabolic Reactions <ul><li>These release energy through the BREAKDOWN of large molecules into smaller units e.g. </li></ul><ul><li>Cellular Respiration: </li></ul><ul><li>ATP -> ADP + Pi </li></ul><ul><li>Also known as exothermic reactions </li></ul>EXOTHERMIC REACTION
  5. 5. Naming Enzymes <ul><li>Enzymes are commonly named by adding a suffix &quot;-ase&quot; to the root name of the substrate molecule it is acting upon. For example, Lipase catalyzes the hydrolysis of a lipid triglyceride. Sucrase catalyzes the hydrolysis of sucrose into glucose and fructose </li></ul><ul><li>A few enzymes discovered before this naming system was devised are known by common names e.g. pepsin, trypsin, and chymotrypsin which catalyse the hydrolysis of proteins </li></ul><ul><li>Enzymes are also given a standard reference number (European Commission Number) to help characterise the 1500 or so enzymes </li></ul>
  6. 6. ENZYMES These catalyse a transfer of a phosphate group onto a molecule such as a carbohydrate or protein Kinases To hydrolyse ATP. Many proteins have an ATPase activity which is essential for their function ATPases To hydrolyse phosphodiester bonds Nucleases To hydrolyse peptide bonds to breakdown proteins -> amino acids Proteases FUNCTION NAME
  7. 7. Form & Function of Enzymes <ul><li>Enzymes work by bringing about substrate(s) of a reaction close together in an active site so that bond breakage or formation occurs at atomic level </li></ul><ul><li>This is often facilitated (helped) by specific chemical effects such as the transfer of proteins or the alteration of charge distribution around the target atoms </li></ul><ul><li>The substrate and enzyme must fit together very precisely </li></ul>
  8. 8. The Catalytic Cycle <ul><li>A cycle of events that describes an enzyme combining with a substrate, remaining unchanged by the reaction and being available at the end of the reaction to combine with another substrate molecule </li></ul>
  9. 9. The Catalytic Cycle of Sucrase <ul><li>Sucrase catalyses the hydrolysis of sucrose into it’s component monosaccharides, GLUCOSE & FRUCTOSE </li></ul><ul><li>1) At the start of the cycle, enzyme ( E ) and substrate ( S ) are available </li></ul><ul><li>2) The molecular interaction of enzyme and substrate at the active site forms the enzyme:substrate complex ( ES ) </li></ul><ul><li>3) Catalysis occurs, forming the enzyme:product complex ( EP ) </li></ul><ul><li>4) Products are released, leaving the enzyme free for the next substrate molecule </li></ul>E S ES EP
  10. 10. Model for Enzyme Action <ul><li>A common model for enzyme action is the lock and key hypothesis </li></ul><ul><li>However, this model is a little misleading in that it tends to give the impression that enzymes are rigid structures, whereas in fact, they are quite flexible and can alter their conformation in response to the binding of other molecules </li></ul><ul><li>The currently accepted model for enzyme action is the INDUCED FIT MODEL , in which conformational changes to the protein occur on binding of a substrate </li></ul>
  11. 11. The Induced Fit Model <ul><li>The enzyme, HEXOKINASE , catalyses the transfer of a phosphate from ATP onto glucose </li></ul><ul><li>The active site and the two domains of the single polypeptide chain are clearly visible in the view of the backbone of the molecule </li></ul><ul><li>Think of the protein about to close around the substrate in the active site similar to the way your hand would close around a door handle </li></ul><ul><li>The effect of this is that glucose fits the active site more closely, and the binding of ATP is also enhanced </li></ul><ul><li>[see diagram of ‘The catalytic cycle of hexokinase] </li></ul>
  12. 12. Control of Enzyme Activity <ul><li>The activity of enzymes must be reguated in some way to avoid metabolic chaos </li></ul><ul><li>Regulation can be achieved through a number of different mechanisms </li></ul><ul><li>A major influence is the NUMBER OF ENZYMES MOLECULES in the cell, which is controlled at the level of gene expression </li></ul><ul><li>COMPARTMENTALISATION also enables the cell to keep sets of enzymes together and away from other enzymes </li></ul><ul><li>TEMPERATURE & pH also affect enzyme activity </li></ul><ul><li>Many enzymes also require CO-FACTORS to function </li></ul>
  13. 13. <ul><li>However, the most effective way of enabling a fine control of enzyme activity is to alter the shape of the enzyme itself, and thus cause a change in its catalytic efficiency </li></ul><ul><li>Examples of this type of metabolic control include INHIBITORS, ALLOSTERIC EFFECTORS , COVALENT MODIFICATION and END-PRODUCT INHIBITION </li></ul>
  14. 14. Inhibitors <ul><li>Enzymes reaction rates can be changed by competitive inhibition and non-competitive inhibition </li></ul><ul><li>Inhibitors can be either competitive or non-competitive </li></ul>
  15. 15. <ul><li>COMPETITIVE inhibitors compete for the active site of the enzyme, thus reducing its effectiveness </li></ul><ul><ul><li>competitive inhibitors are usually similar in structure to the substrate and the enzyme is ‘fooled’ into accepting the inhibitor, which blocks the active site </li></ul></ul>
  16. 16. <ul><li>E.G: An example for competitive inhibition is the enzyme succinate dehydrogenase by malonate . Succinate dehydrogenase catalyses the oxidation of succinate to fumarate . </li></ul>
  17. 17. <ul><li>NON-COMPETITIVE inhibitors bind at a different location and change the conformation of the enzyme, thus altering the shape of the active site and again reducing the catalytic efficiency </li></ul>
  18. 18. <ul><li>Inhibition can either be reversible or non-reversible depending on how the inhibitor binds to the enzyme </li></ul><ul><li>Some inhibitors bind irreversibly with the enzyme molecules, inhibiting the catalytic activities permanently. The enzymatic reactions will stop sooner or later and are not affected by an increase in substrate concentration. These are irreversible inhibitors . </li></ul><ul><li>Examples are heavy metal ions including silver , mercury and lead ions. </li></ul>
  19. 19. Allosteric Enzymes <ul><li>These are enzymes that ‘change shape’ in response to the binding of a regulating molecule (often called a modulator or effector ) </li></ul><ul><li>Allosteric modulators can be either positive or negative effectors of enzyme activity </li></ul><ul><li>They function by binding to allosteric sites that are distinct from the active site of the enzyme </li></ul><ul><li>Non-competitive inhibition is a form of allosteric regulation </li></ul>
  20. 20. Allosteric Enzymes
  21. 21. <ul><li>In multi-subunit enzymes, the structure is more complex, and the enzyme often exists in 2 different conformational states: </li></ul><ul><ul><li>ACTIVE and INACTIVE </li></ul></ul><ul><li>These can be stabilised by binding the modulator </li></ul><ul><ul><li>Positive modulators stabilise the active form of the enzyme </li></ul></ul><ul><ul><li>Negative modulators stabilise the inactive form </li></ul></ul><ul><li>In addition to these modulators changing the activity of allosteric enzymes, sometimes the binding of the substrate itself to one active site enhances binding at the other active sites. This is known as COOPERATIVITY </li></ul>
  22. 22. Covalent Modification <ul><li>Covalent modification of enzymes is another strategy used widely in metabolic regulation </li></ul><ul><li>One of the most common modifications is the addition of a PHOSPHATE group, which can alter the shape of a protein by attracting positively charged R-groups [phosphates carry 2 negative charges on the 2 single-bonded O atoms] </li></ul><ul><li>PROTEIN KINASES add phosphate groups and PHOSPHATASES remove them, thus the effect can be REVERSED </li></ul><ul><li>Some proteins are activated by phosphorylation , others are inactivated </li></ul>
  23. 23. <ul><li>An example of phosphorylation activating an enzyme is the skeletal muscle enzyme GLYCOGEN PHOSPHORYLASE </li></ul><ul><li>This enzyme releases glucose molecules from glycogen when heavy demands are placed on muscle tissue </li></ul><ul><li>This process is highly regulated. Traffic of sugar into and out of storage in glycogen is used to control the level of glucose in the blood, so glycogen phosphorylase must be activated when sugar is needed and quickly deactivated when glucose is plentiful </li></ul>
  24. 24. <ul><li>Glycogen phosphorylase is present as an inactive non-phosphorylated form which is converted to the active phosphorylated form by the addition of a phosphate group to a serine residue in the protein by the enzyme PHOSPHORYLASE KINASE </li></ul><ul><li>When the demand for glucose drops, PHOSPHORYLASE PHOSPHATASE removes the phosphate group and inactivates the enzyme </li></ul>However … glycogen phosphorylase is also regulated by an allosteric effect !
  25. 26. <ul><li>Glucose and ATP act as negative modulators and AMP (adenine monophosphate) acts as a positive modulator – also causing the enzyme to shift to the active conformation </li></ul><ul><li>This is useful, because AMP is a product of ATP breakdown and will be more plentiful when energy levels are low and more glucose is needed </li></ul><ul><li>A further complication is that there is a hormonal control mechanism by adrenaline and glucagon </li></ul>
  26. 27. Proteolytic Cleavage <ul><li>Another form of control by a covalent activating mechanism is proteolytic cleavage as found in the enzyme TRYPSIN </li></ul><ul><li>Trypsin is synthesised in the pancreas, but not in its active form as it would digest the pancreatic tissue </li></ul><ul><li>Therefore it is synthesised as a slightly longer protein called TRYPSINOGEN , which is inactive </li></ul><ul><li>Activation occurs when trypsinogen is cleaved by a protease in the duodenum </li></ul><ul><li>Once active, trypsin can activate more trypsinogen molecules, resulting in an autocatalytic cascade that produces a large number of active trypsin molecules very rapidly </li></ul>
  27. 28. End-Product Inhibition <ul><li>Metabolism is organised as a series of metabolic pathways, and control of these pathways is an important feature of cell biochemistry </li></ul><ul><li>One way in which control can be exercised is END-PRODUCT INHIBITION </li></ul><ul><li>End-product inhibition is energetically efficient as it avoids the excessive (and wasteful) production of the intermediates of a pathway </li></ul><ul><li>This is a form of NEGATIVE FEEDBACK </li></ul>
  28. 29. For example, in the production of the amino acid isoleucine in bacteria, the initial substrate is threonine which is converted by five intermediate steps to isoleucine. As isoleucine begins to accumulate, it binds to an allosteric site of the first enzyme in the pathway thereby slowing down its own production. In this way, the cell does not produce any more isoleucine than is necessary.