1. Control of Metabolism Chapter 4

2. Topics 1.Overview of metabolic control at various level 2.Enzyme reactions and cofactors 3.Regulation of enzyme activities

3. 4.1: Overview of metabolic control at various levels Environment around bacteria is constantly changing. So they adapt to limitations They developed complex mechanism for regulation of catabolic and anabolic pathways

4. Bacteria exert control over their metabolism at every possible stage. Starting at the level of the gene that encodes for a protein and ending with alteration or modifications in the protein after it is produced.

5. Points for regulation of various metabolic processes.

6. Conditions Affecting Enzyme Formation in Bacteria Bacterial cells can change patterns of enzymes to adapt to their specific environment. The concentration of an enzyme in a bacterial cell depends on the presence of the substrate for the enzyme. Constitutive enzymes are always produced by cells independently of the composition of the medium in which the cells are grown. Example: The enzymes that operate during glycolysis and the TCA cycle. They are present at around the same concentration in cells at all times.

7. Inducible enzymes are produced "turned on" in cells in response to a particular substrate. They are produced only when needed. In the process of induction, the substrate, or a compound structurally similar to the substrate, evokes formation of the enzyme and is called an inducer. Repressible enzymes synthesis are regulated or "turned off" by the presence of (for example) the end product of a pathway that the enzyme normally participates in. In this case, the end product is called a co-repressor of the enzyme.

8. 4.2: Enzyme reactions and cofactors Regulatory enzymes are usually the enzymes that are the rate-limiting, or committed step, in a pathway, meaning that after this step a particular reaction pathway will go to completion.

9. In bacterial cells, enzymatic reactions may be regulated by two unrelated modes: 1.Control or regulation of enzyme activity (feedback inhibition or end product inhibition), which mainly operates to regulate biosynthetic pathways; and 2.Control or regulation of enzyme synthesis, including end-product repression, which functions in the regulation of biosynthetic pathways, and enzyme induction and catabolite repression, which regulate mainly degradative pathways.

10. Properties of Enzymes Biological catalysts Not consumed during a chemical reaction Speed up reactions from 1000 - 10 20, with a mean increase in rate of 100,000 Exhibit stereospecificity  act on a single stereoisomer of a substrate Exhibit reaction specificity  no waste or side reactions

11. Enzymes provide a surface on which reactions take place Active site: the area on the enzyme surface where the enzyme forms a loose association with the substrate Substrate: the substance on which the enzyme acts Enzyme-substrate complex: formed when the substrate molecule collides with the active site of its enzyme Endoenzymes (intracellular)/exoenzymes (extracellular) Activation energy: the minimum energy required to start a chemical reaction Transition state – the intermediate stage in a reaction in which the old bonds break and new bonds are formed

12. Nomenclature Typically add “-ase” to name of substrate e.g. lactase breaks down lactose (dissacharide of glucose and galactose)

13. Classifications IUBMB classifies enzymes based upon the class of organic chemical reaction catalyzed: 1.Oxidoreductase - catalyze redox reactions; dehydrogenases, oxidases, peroxidases, reductases 2.Transferases - catalyze group transfer reactions; often require coenzymes 3.Hydrolases - catalyze hydrolysis reactions 4.Lyases - lysis of substrate; produce contains double bond 5.Isomerases - catalyze structural changes; isomerization 6.Ligases - ligation or joining of two substrates with input of energy, usually from ATP hydrolysis; often called synthetases or synthases

14. Figure 5.2 Energy Requirements of a Chemical Reaction

15. Enzyme Components Apoenzyme: Protein Holoenzyme: Apoenzyme plus cofactor Cofactor: Nonprotein component (e.g. magnesium, zinc) Coenzyme: Organic cofactor (Eg: NADH, FADH) Many enzymes can catalyze a reaction only if coenzymes, or cofactors are present.

16. Figure 5.3 Components of a Holoenzyme

17. The Parts of an Enzyme

18. Coenzymes Many are derived from vitamins. Eg: 1.Niacin NAD (Nicotinamide adenine dinucleotide) 2.Riboflavin FAD (Flavin adenine dinucleotide) 3.Pantothenic Acid CoEnzyme A

19. Mechanism 1.Substrate binding 2.Formation enzyme substrate complex 3.Production formation and dissociation 4.Enzyme recovery

20. Figure 5.4a The Mechanism of Enzymatic Action

21. Each substrate binds to an active site, producing an enzyme-substrate complex. The enzyme helps a chemical reaction occur, and one or more products are formed

22. Factors Influencing Enzyme Activity 1.Temperature 2.pH 3.Substrate concentration 4.Enzyme concentration 5.Inhibitors

23. Temperature Enzymes are affected by heat Most enzymes have an optimum temperature, near normal body temperature at which they catalyze a reaction most rapidly The rate at which an enzyme catalyzes a reaction increases with temperature up to the optimum T

24. Figure 5.5a Effect of Temperature on Enzyme Activity

25. pH Enzymes are affected by extremes of pH Even small pH changes can alter the electrical charges on various chemical groups in enzyme molecules, thereby altering the enzyme’s ability to bind its substrate and catalyze a reaction Enzymes catalyze a reaction most rapidly at an optimum pH, near neutral,

26. Figure 5.5b Effect of pH on Enzyme Activity

27. Substrate concentration Increasing the [substrate] increases the rate of reaction (enzyme activity). enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction rate will plateau.

28. Figure 5.5c Effect of Substrate Concentration on Enzyme Activity

29. Enzyme concentration The higher the concentration of an enzyme the greater should be the initial reaction rate This will last as long as substrate present

30. Enzyme Inhibition Competitive inhibitor: A molecule similar in structure to a substrate can bind to an enzyme’s active site and compete with substrate Noncompetitive inhibitors: attach to the enzyme at an allosteric site, which is a site other than the active site distort the tertiary protein structure and alter the shape of the active site Feedback inhibition: regulates the rate of many metabolic pathways when an end product of a pathway accumulates and binds to and inactivates the first enzyme in the metabolic pathway

31. Figure 5.7a–b Enzyme Inhibitors: Competitive Inhibition

32. Competitive inhibition of enzymes

33. Figure 5.7a, c Enzyme Inhibitors: Noncompetitive Inhibition

34. Noncompetitive (allosteric) inhibition of enzymes

35. 4.3: Regulation of enzyme activities Mechanisms 1.Enzyme modification 2.Enzyme denaturation 3.Isoenzyme regulation 4.Allosteric binding 5.Feedback inhibition

36. Enzymes modification Can either activate it or inhibit it by altering the conformation of the enzyme or by serving as a functional group in the active site

37. Enzyme denaturation

38. Enzyme denaturation (cont’)

39. Isoenzyme regulation Enzymes that catalyze the same reaction (catalytically and structurally similar) but are encoded by different genes Glycogen phosphorylase-synthesize in liver, brain and muscle-involves in degradation of glycogen Isoenzymes = isoforms

40. Allosteric regulation of enzyme activity Allosteric enzyme: Allosteric enzyme: an oligomer whose biological activity is affected by other substances binding to it these substances change the enzyme’s activity by altering the conformation(s) of its 4°structure Allosteric regulators generally act by increasing or decreasing the enzyme’s affinity for the substrate

41. The key to allosteric behavior is the existence of multiple forms for the 4°structure of the enzyme allosteric effector: allosteric effector: a substance that modifies the 4° structure of an allosteric enzyme homotropic effects: homotropic effects: allosteric interactions that occur when several identical molecules are bound to the protein. heterotropic effects: heterotropic effects: allosteric interactions that occur when different substances are bound to the protein.

42. A change in conformational structure at one location of a multisubunit protein that causes a conformational change at another location on the protein Effectors – i) serves as stimulants to enzyme (+ve effectors) = increase catalytic activity – ii) inhibitors (-ve effectors) to enzyme = reduce/inhibit catalytic activity - Act by reversible, noncovalent binding to a site on the enzyme

43. Homotropic allosterism

44. Heterotrophic allosterism

45. Enzyme inhibition: Feedback inhibition Product (usually ultimate product) of a pathway controls the rate of synthesis through inhibition of an early step (usually the first step) to conserve material and energy by preventing accumulation of intermediates

46. Figure 5.8 Enzyme Inhibitors: Feedback Inhibition

47. End of chapter