Ch. 19 - Enzymes and Vitamins

1. Ch. 19 - Enzymes and Vitamins • Living systems are shaped by an enormous variety of biochemical reactions nearly all of which are mediated by a series of remarkable biological catalysts known as enzymes. • Enzymology (study of enzymes) and biochemistry evolved together from the 19th century investigation of fermentation. • “enzyme” – Greek: en, in + zyme, yeast • E. Buchner showed that alcohol fermentation (ethanol production from glucose) could be carried out using cell-free yeast extract.

2. What are enzymes? • Enzymes are biological catalysts. • Would you expect enzymes to be fibrous or globular proteins? • They are extremely effective, increasing reaction rates from 106 to 1012 times. • Most enzymes act specifically with only one reactant (called a substrate) to produce products

3. Enzymes facillitate chemical reactions in an active site, a pocket in an enzyme with the specific shape and chemical makeup necessary to bind a substrate and where the reaction takes place. The amino acids His, Cys, Asp, Arg, and Glu participate in 65% of all active sites. • A living cell has a set of some 3,000 enzymes that it is genetically programmed to produce. If even one enzyme is missing or defective, the results can be disastrous. • Enzymes are used in household products including meat tenderizer, facial treatments, laundry products, and contact lens cleaners.

4. An example of an enzyme found in many living organisms is catalase 2 H2O2 (l)  2 H2O (l) + O2(g) Each molecule of catalase is a tetramer of four polypeptide chains. Each chain is composed of more than 500 amino acids. Located within this tetramer are four porphyrin heme groups much like the familiar hemoglobins, cytochromes, chlorophylls and nitrogen-fixing enzymes in legumes. The heme group is responsible for catalase’s enzymatic activity.

5. What are some general characteristics of an enzyme? • • Specificity • Enzymes have a great degree of specificity with respect to the identities of the substrate (reactants) and their products than do chemical catalysts. • Absolute specificity: • Enzymes catalyzing of the reaction of one and only one substance • Relative specificity: • Enzymes that catalyze the reaction of several structurally related substances • Stereochemical specificity: • Enzymes that are able to distinguish between stereoisomers

6. The specificity of an enzyme for one of two enantiomers is a matter of fit. One enantiomer fits better into the active site of the enzyme than the other enantiomer. An enzyme catalyzes reaction of the enantiomer that fits better into the active site of the enzyme.

7. Muscle Relaxants and Enzyme Specificity The mode of action of succinylcholine, a muscle relaxant used during minor surgery, utilizes the substrate specificity of the enzyme, acetylcholinesterase. Acetylcholine Succinylcholine

8. • Catalytic Efficiency • Rates of enzymatically catalyzed reactions are typically 106 to 1012 times greater than those of the corresponding uncatalyzed reactions • Turnover number: The number of molecules of substrate acted on by one molecule of enzyme per minute • Ex: Carbonic anhydrase converts carbon dioxide to bicarbonate at a rate of 36 million molecules per minute. CO2 + H2O HCO3- + H+

9. • Milder Reaction Conditions • Reactions occur under relatively mild conditions • Temperature below 100°C, atmospheric pressure, and nearly neutral pH

10. • Cofactors • Many enzymes are conjugated proteins that require non-protein portions known as cofactors. • Some cofactors are metal ions. Carboxypetptidase is an enzyme that requires a Zn 2+ ion as a cofactor. This enzyme hydrolyzes the first amide bond at the C-terminal end of peptides. Carboxypeptidase is synthesized in the pancreas and secreted in the small intestine. A “space-filled” representation of carboxypeptidase

11. A proposed model of the active-site chemistry of carboxypeptidase

12. Others cofactors are non-protein organic molecules called coenzymes. Lactate dehydrogenase is an enzyme that requires the coenzyme NAD+ /NADH for enzymatic function. Which species have been reduced? Oxidized?

13. Remember, enzymes catalyze both the forward and the reverse processes. Biochemical reactions are often represented with the coenzymes/ enzymes written in conjuction with the equation “reacts to form” arrow.

14. Structure of NAD+ and NADH NAD+ Nicotinamide adenine dinucleotide NADH Nicotinamide adenine dinucleotide

15. • Capacity for Regulation • Cells control the rates of reactions and the amount of any given product formed by regulating the action of the enzyme. • Mechanisms for regulatory process: • Allosteric and feedback control • Covalent modification of the enzyme • Variation of enzyme concentration

16. How are enzymes classified? Enzymes may be classified according to the type of reaction that they catalyze. Six main classes: 1. Oxidoreductase: redox reactions 2. Transferase: transfer functional groups 3. Hydrolase: hydrolysis reactions 4. Lyase: addition and elimination reactions 5. Isomerase: isomerization reaction 6. Ligase: bond formation coupled with ATP

17. Oxidoreductases • Catalyze oxidation-reduction (redox) reactions, most commonly addition or removal of oxygen or hydrogen. • Requires coenzyme

18. Transferases • Catalyze transfer of a functional group from one molecule to another • Kinase applied to enzymes that catalyze transfer of terminal phosphate group

19. Hydrolases • Catalyze the hydrolysis of substrate – the breaking of bond with addition of water. • These reactions are important in the digestive process.

20. Isomerases • Catalyze the isomerization of a substrate in reactions that have one substrate and one product • Rearranges of the functional groups within a molecule (catalyst converts one isomer in to the other)

21. Lyases • Catalyze the addition of groups such as H2O, CO2, or NH3 to a double bond or reverse reaction in which a molecule is eliminated to create a double bond

22. Ligases • Catalyze the bonding of two substrate molecules • reaction where C-C, C-S, C-O, or C-N bond is made or broken • Accompanied by ATP-ADP conversion (release of energy drives reaction)

23. Oxidoreductase Oxidase Oxidation of a substrate Reductase Reduction of a substrate Dehydrogenase: Introduction of a double bond (C-C or C-O) Transferase Transaminase Transfer amino groups Kinase Transfers a phosphate group Hydrolyase Lipase Hydrolyzes ester groups of lipids Protease Hydrolyzes amide bonds of proteins Nuclease Hydrolyzes phosphate esters in nucleic acids Lyase Dehydrase Loss of water from a substrate Decarboxylase Loss of carbon dioxide from a substrate Ligase Synthetase Formation of a new C-C bond from two substrates Carboxylase Formation of a new C-C bond with carbon dioxide. Isomerase Epimerase Isomerization of a chiral carboncenter Subclasses and Types of Reaction

24. How are enzymes named? Common Names Derived from the name of the substrate Urease Lactase Derived from the reactions they catalyze Dehydrogenase Decarboxylase Historical Names Have no relationship to either substrate or reaction catalase, pepsin

25. Systematic Naming • Unambiguous (often very long) • Specifies: • Substrate (substance acted on) • Functional group • Type of reaction catalyzed • Names end in –ase

26. Example • Systematic Name: Urea amidohydrolase • Substrate: urease • Functional group: amide • Type of reaction: hydrolysis • Common name: Urease

27. Can you name the missing enzymes?

28. How do enzymes work? • General Theory • Substrate and enzyme molecules come into contact and interact over only a small region of the enzyme surface (active site) • The substrate bonds to active site via temporay non-covalent interactions and covalent interactions (less frequently). • An enzyme-substrate complex (ES) is formed when a substrate and enzyme bond. • The substrate is destabilzed in the ES complex by various non-covalent forces.

29. Two models are proposed to represent the interaction between substrates and enzymes. These are: Lock-and-key model: The substrate is described as fitting into the active site as a key fits into a lock. Induced-fit-model: The enzyme has a flexible active site that changes shape to accommodate the substrate and facilitate the reaction.

32. Mechanisms of Catalysis The chemistry at the active site of an enzyme is the most important factor in catalytic effect of an enzyme. • Proximity Effect – the enzyme positions the reactant(s) for a reaction • Orientation Effect – positioning of the reactant(s) in the active site allows for optimum orientation • Catalytic Effect – atoms at the active site provide structural features that facillitate the chemistry of the reaction • Energy Effect –the activation energy requirements for the reaction are reduced due to any combination of the above

33. Hydrolysis of a peptide bond by chymotrypsin

34. How is enzyme activity measured? • Experiments that measure enzyme activity are called assays. • Assays for blood enzymes are performed in medical laboratories • Some assays determine how fast the characteristic color of a product forms or the color of a substrate decreases • Some assays are based on reactions in which protons are produced or used up. This type of enzyme activity can be followed by measuring how fast the pH of the reacting mixture changes with time.

35. Diagnostically Useful Assays • Alanine Transamininase (AST) • Hepatitis • Lactate dehydrogenase (LDH) • Heart attacks, liver damage • Acid phosphatase • Prostate cancer • Creatine Kinase (CK) • Heart attacks

37. What are some factors that effect the rate of enzyme catalyzed reactions? • Enzyme Concentration • Substrate Concentration • Temperature • pH

38. Effect of Concentration • Substrate concentration: At low substrate concentration, the reaction rate is directly proportional to the substrate concentration. With increasing substrate concentration, the rate drops off as more of the active sites are occupied. • Enzyme concentration: The reaction rate varies directly with the enzyme concentration as long as the substrate concentration does not become a limitation.

39. Change of reaction rate with substrate concentration when enzyme concentration is constant.

40. Change of reaction rate with enzyme concentration with no limit on the substrate concentration.

41. Effect of Temperature and pH • Effect of Temperature: Increase in temperature increases the rate of enzyme catalyzed reactions. The rates reach a maximum and then begins to decrease. The decrease in rate at higher temperature is due to denaturation of enzymes.

42. Effect of temperature on reaction rate

43. Effect of pH: The catalytic activity of enzymes depends on pH and usually has a well defined optimum point for maximum catalytic activity.

44. How is enzyme activity regulated? • We will see that there are several modes of enzyme regulation: • • Feedback • • Allosterism • • Inhibition • • Covalent Modification and Genetic Control • Any process that starts or increase the activity of an enzyme is activation. • Any process that stops or slows the activity of an enzyme is inhibition.

45. Two of the mechanisms that control the enzymes activity are: • Feedback control: Regulation of an enzyme’s activity by the product of a reaction later in a pathway.

46. Example of Feedback Control: Synthesis of Isoleucine • Threonine deaminase (enzyme that catalyzes 1st step) is subject to inhibition by the final product (isoleucine) • Isoleucine binds to a different site on the enzyme and changes the conformation and threonine binds poorly • As the concentration of isoleucine increases, the enzyme activity drops.

47. Allosteric control: Activity of an enzyme is controlled by the binding of an activator or inhibitor at a location other than the active site. • Positive allosteric regulator - changes the active site making the enzyme a better catalyst (rate accelerates). • Negative allosteric regulator - changes the active site so that the enzyme becomes a less effective catalyst (rate decreases).

48. A positive regulator changes the activity site so that the enzyme becomes a better catalyst and rate accelerates. A negative regulator changes the activity site so that the enzyme becomes less effective catalyst and rate slows down.

49. Covalent modification Enzyme availability can be controlled by storing the enzyme in its inactive form called zymogens or proenzymes. When the active enzyme is needed, the stored zymogen is released from storage and activated at the location of the reaction. Activation of a zymogen requires a covalent modication of its structure. Examples: Pepsinogen  pepsin Digestion of proteins Prothrombin  thrombin Blood clotting