2. Enzyme

3. Why Are Enzymes So Important? Why are we devoting two whole lecture topic to a enzyme? Nearly all chemical reactions in biological cells need enzymes to make the reaction occur fast enough to support life. Image: Jumping rope, Meagan E. KleinJumping rope From the Virtual Cell Biology Classroom on ScienceProfOnline.comVirtual Cell Biology ClassroomScienceProfOnline.com

4. Composition, structure and properties of enzyme How Enzymes work Enzyme activity Factors affecting enzyme activity Regulation of enzyme activities Enzymes in clinical diagnosis Outline

5. 1. Definition of enzyme Enzymes are biological catalysts. A Catalyst is defined as "a substance that increases the rate of a chemical reaction without being itself changed in the process.”

6. Enzymes as Biological Catalysts Enzymes are proteins that increase the rate of reaction by lowering the energy of activation They catalyze nearly all the chemical reactions taking place in the cells of the body Enzymes have unique three-dimensional shapes that fit the shapes of reactants (substrates)

7. 2. Properties of enzymes (important!) Catalytic efficiency – high efficiency, 10 3 to 10 17 faster than the corresponding uncatalyzed reactions Specificity - high specificity, interacting with one or a few specific substrates and catalyzing only one type of chemical reaction. Mild reaction conditions- 37 ℃, physiological pH, ambient atmospheric pressure

8. 3. Chemical composition of enzymes (1) Simple protein (2) Conjugated protein Holoenzyme= Apoenzyme+ Cofactor Coenzyme : loosely bound to enzyme (non- covalently bound). Prosthetic group : very tightly or even covalently bound to enzyme (covalently bound) Cofactor

10. 4. Classification of enzymes 1). Monomeric enzyme 2). Oligomeric enzyme 3). Multienzyme complex: such as Fatty acid synthase (1). By their composition

11. (2) Nomenclature Recommended name Enzymes are usually named according to the reaction they carry out. To generate the name of an enzyme, the suffix -ase is added to the name of its substrate (e.g., lactase is the enzyme that cleaves lactose) or the type of reaction (e.g., DNA polymerase forms DNA polymers). Systematic name (International classification) By the reactions they catalyze (Six classes)

12. 5. How enzymes work (important!) 1) Enzymes lower a reaction’s activation energy –All chemical reactions have an energy barrier, called the activation energy, separating the reactants and the products. –activation energy: amount of energy needed to disrupt stable molecule so that reaction can take place.

13. Enzymes Lower a Reaction’s Activation Energy

14. What is the difference between an enzyme and a protein? Protein All enzymes are proteins except some RNAs not all proteins are enzymes RNA Enzymes

15. 2) The active site of the enzyme Enzymes bind substrates to their active site and stabilize the transition state of the reaction. The active site of the enzyme is the place where the substrate binds and at which catalysis occurs. The active site binds the substrate, forming an enzyme-substrate(ES) complex. Active site Binding site Catalytic site

16. Enzymatic reaction steps 1. Substrate approaches active site 2. Enzyme-substrate complex forms 3. Substrate transformed into products 4. Products released 5. Enzyme recycled

17. 6. Enzyme activity Enzymes are never expressed in terms of their concentration (as mg or μg etc.), but are expressed only as activities. Enzyme activity = moles of substrate converted to product per unit time. –The rate of appearance of product or the rate of disappearance of substrate –Test the absorbance: spectrophotometer

18. 7. Factors affecting enzyme activity Concentration of substrate Concentration of enzyme Temperature pH Activators Inhibitors

19. Enzyme velocity Enzyme activity is commonly expressed by the intial rate (V 0 ) of the reaction being catalyzed. (why?) Enzyme activity = moles of substrate converted to product per unit time.

20. Note: V means V 0 [S] [S] + K M V = V max K m : Michaelis constant K m = (k 2 + k 3 )/k 1 Michaelis-Menten equation (very important!) 1. Michaelis-Menten equation describes how reaction velocity (V) varies with substrate concentration [S]. The following equation is obtained after suitable algebraic manipulation.

21. [S]>>[E]V ∝ [E] The initial rate of an enzyme-catalyzed reaction is always proportionate to the concentration of enzyme. This property of enzyme is made use in determining the serum enzyme for the diagnosis of diseases. (2) Effect of [E] on velocity

22. (3) Effect of temperature on velocity Bell-shaped curve

23. (4) Effect of pH value on velocity Each enzyme has an optimal pH or pH range (where the enzyme has maximal activity). Requirements for the catalytic groups in the active site in appropriate ionization state is a common reason for this phenomenon. The pH optimum varies for different enzymes. Most enzyme: neutral pH (6- 8). Bell-shaped curve

24. (5) Effect of activator on velocity (i). Inorganic ions Metal ions ， such as Na +, K +, Mg 2+, Ca 2+, Cu 2+, Zn 2+, Fe 2+ et al Anions: such as Cl -, Br -, I - 、 CN - et al (ii). Organic (iii). Proteins Reducing agents, such as Cys 、 GSH Enzyme activators are molecules that bind to enzymes and increase their activity.

25. (6) Inhibition of enzyme activities (very important!) Inhibitor: any molecule which acts directly on an enzyme to lower its catalytic rate is called an inhibitor.(not denaturation) Some enzyme inhibitors are normal body metabolites. Other may be foreign substances,such as drugs or toxins.

26. 8. REGULATION OF ENZYME ACTIVITY 1.Allosteric binding sites: Allosteric enzymes are regulated by molecules called effectors (modifiers) that binds nonconvalently at a site other than the active site. 2. By Covalent Modification: Many enzymes are regulated by covalent modification, most frequently by the addition or removal of ‘phosphate’ group to serine, threonine or tyrosine residue of the enzyme by kinases. (enzyme) 3.Induction and repression of enzyme sysnthesis: Cells can also regulate the amount of enzymes present by altering the rate of enzyme synthesis.

27. REGULATION CONT…. 4. Zymogen Cleavage: Some enzyme are synthesized as inactive precursor, called zymogens, that are activated by proteolysis (e.g., digestive enzyme, pepsinogen is inactive and cleaved to pepsin which is active chymotrypsin) 5.Location within the cell: Many enzymes are localized in specific organelles within the cell. This, compartmentation helps in the regulation of the metabolic pathway.

28. 9. Enzymes in clinical diagnosis An enzyme test is a blood test or urine test that measures levels of certain enzymes to assess how well the body’s systems are functioning and whether there has been any tissue damage. (why?)

29. Common enzymes used for clinical diagnosis include: –a–alanine aminotransferase(ALT,also called glutamate pyruvate transaminase,GPT) –a–alkaline phosphatase –a–amylase –a–aspartate aminotransferase –c–creatine kinase –l–lactate dehydrogenase