POINT- BIOCHEMISTRY SEM 2 ENZYMES UNIT 5.pptx

BIOCHEMISTRY

1. E N Z YM E FP P T . c om

2. S yllabus

3. I NT R O D U C T I ON E n z ym e s 3

4. I NT R O D U C T I ON 4 Enzymes are biocatalysts – the catalysts of life. A catalyst is defined as a substance that increases the velocity or rate of a chemical reaction without itself undergoing any change in the overall process. Enzymes may be defined as biocatalysts synthesized by living cells.

5. 5 PROPERTIES

6. 6 PROPERTIES They are protein in nature colloidal thermolabile in character specific in their action.

7. PROPERTIES The functional unit of the enzyme is known as holoenzyme Holoenzyme made up of apoenzyme (the protein part) and a coenz ym e (n o n - p r o t e i n o r ga n i c part). Holoenzyme (ac ti v e en z ym e ) Apoenzyme + Coenzyme (protein part) (non-protein part) 9

8. S TRUCTURE OF E NZYMES the region t h a t b i nd s The active site of an enzyme is s ub s t r a t e s , c o - f a c t o r s an d p r o s t he t i c g r oup s an d c on t a i n s Active site has a specific protein. s hap e due to tertiary structure of A change in the shape of protein affects the shape of active site and function of the enzyme.

9. A CTIVE SITE Bindi n g Si te It chooses the substrate and binds it to active site . Cata l y t i c Si t e It performs the catalytic action of enzyme. o Active site can be further divided into: Active Site

10. CO-FACTORS

11. I NORGANIC CO - FACTORS o These are the inorganic molecules required for the proper activity of enzymes. O RGANIC CO - FACTORS o These are the organic molecules required for the proper activity of enzymes.

12. TYPES OF O RGANIC CO - FACTORS Prosthetic Group Coenzyme o A pro s th e tic tightly bound g r ou p is a o A c oen z y me is organic co- b o u n d org a n i c loosely co-facto + r. E.g. NAD + factor e.g . F l a v in s , heme groups and biotin .

13. SUBSTRATE The reactant in biochemical reaction is termed as substrate. When a substrate binds to an enzyme it forms an enzyme- substrate complex. Enzyme J o ins Substrate

14. CLASS Enzymes are sometimes considered under two broad categories : (a) Intracellular enzymes – They are functional within cells where they are synthesized. (b) Extracellular enzymes – These enzymes are active outside the cell; all the digestive enzymes belong to this group.

15. N OMENCLATURE OF ENZYMES o An enzyme is named according to the name of the substrate it catalyses. o S om e en z y m e s w ere name d be f o r e a s y s t ema t i c naming enzyme was formed. Example : pepsin, trypsin and rennin w a y of B y add i n g s uff i x -ase a t t h e en d o f t h e nam e of t h e substrate, enzymes are named. Enzyme for catalyzing the hydrolysis is termed as hydrolase.

16. E XAMPLE substrate enzymes products lactose lact ase g l uco s e + ga l ac to se maltose malt ase Glucose cellulose cellul ase Glucose lipid lip ase G l yce ro l + f a t t y ac i d starch amyl ase Maltose protein prote ase Peptides + polypeptide

17. C LASSIFICATION The International Union of Biochemistry (IUB) appointed an Enzyme Commission in 1961. Since 1964, the IUB system of enzyme classification has been in force. Enzymes are divided into six major classes (in that order). Each class on its own represents the general type of reaction brought about by the enzymes of that class

18. [The word OTHLIL (first letter in each class) may be memorised to remember the six classes of enzymes in the correct order]. Each class in turn is subdivided into many sub- classes which are further divided. A four digit Enzyme Commission (E.C.) number is assigned to each enzyme representing the class (first digit), sub-class (second digit), sub-sub class (third digit) and the individual enzyme (fourth digit). Each enzyme is given a specific name indicating the substrate, coenzyme (if a n y ) and the type o f the r eact i o n c a t a l y se d b y the e n z ym e .

19. The contact between the enzyme and substrate is the most essential pre-requisite for enzyme activity. Factors affecting Enzyme

20. Factors affecting Enzyme

21. 1. Concentration of enzyme As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases Factors affecting Enzyme

22. 2. Concentration of substrate Increase in the substrate concentration gradually increases the velocity of enzyme reaction within the limited range of substrate levels. A rectangular hyperbola is obtained when velocity is plotted against the substrate concentration . Factors affecting Enzyme

23. 3. Effect of temperature Velocity of an enzyme reaction increases with increase temperature up to a maximum and then declines. A bell-shaped curve is usually observed . T e m pe r a t u r e c o e f f ic i e n t or Q 1 is d e fi n e d as in c r ease in enzyme velocity when the temperature is increased by 10°C. Factors affecting Enzyme

24. 4. Effect of pH Increase in the hydrogen ion concentration (pH) considerably i n f l u e nc es t h e e n z y m e a c tivity a n d a bell - s hap e d cu r v e is normally obtained E a c h enz y m e has an op t imum pH at which the velocity is maximum. Below and above this pH, the enzyme activity is much lower and at extreme pH, the enzyme becomes totally inactive. Factors affecting Enzyme

25. 5. Effect of product concentration The accumulation of reaction products generally decreases the enzyme velocity. For certain enzymes, the products combine with the active site of enzyme and form a loose complex and, thus, inhibit the enzyme activity. Factors affecting Enzyme

26. 6. Effect of activators Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, K+ etc. for theiroptimum activity. Rarely, anions are also needed For enzyme activity e.g. chloride ion Two categories of enzymes requiring metals for their activity are distinguished 1. Metal-activated enzymes : The metal is not tightly held by the enzyme and can be exchanged easily with other ions e.g. ATPase (Mg2+ and Ca2+) Enolase (Mg2+) 2.Metalloenzymes : These enzymes hold the metals rather tightly which are not readily exchanged. Factors affecting Enzyme

27. 7. Effect of time Under ideal and optimal conditions (like pH, temperature etc.), the time required for an enzyme reaction is less. Variations in the time of the reaction are generally related to the alterations in pH and temperature. Factors affecting Enzyme

28. 8. Effect of light and radiation Exposure of enzymes to ultraviolet, beta, gamma and X- rays inactivates certain enzymes due to the formation of peroxides. e.g. UV rays inhibit salivary amylase activity. Factors affecting Enzyme

29. Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction is investigated. Studying an enzymes kinetics in this way can reveal the catalytic mechanism of this enzyme

30. Km value is a constant and a characteristic feature of a given enzyme . A low Km value indicates a strong affinity between enzyme and substrate, whereas a high Km value reflects a weak affinity between them. For majority of enzymes, the Km values are in the range of 10–5 to 10–2 moles. It may however, be noted that Km is not dependent on the concentration of enzyme.

31. L i n e w ea v e r - B u r k d o u b l e r e c i p r o c a l p l o t : For the determination of Km value, the substrate saturation curve ( Fig.) is not very accurate since Vmax is approached asymptotically. By taking the reciprocals o f the equ a tion (1), a straight line graphic representation is obtained.

32. L i n e w ea v e r - B u r k d o u b l e r e c i p r o c a l p l o t :

33. REGULATION OF ENZYME Enzyme regulation definition: “Process, by which cells can turn on, turn off, or modulate the activities of various metabolic pathways by regulating the activity of enzyme ”

34. WHAT DO YOU UNDESTAND BY R E G U L A T I O N O F E NZ Y M E

35. Enzyme regulation is the control of the rate of a reaction catalyzed by an enzyme by some effector (e.g., inhibitors or activators ) or by alteration of some condition ( ph or ionic strength )

36. There are many kinds of molecules that block or promote enzyme function, and that affect enzyme function by different routes.

37. ENZYME INHIBITORS WITH EXAMPLES An enzyme inhibitor is a molecule/substance that binds to an enzyme and blocks its activity.

38. TYPES Types of enzyme inhibition: competitive, noncompetitive, and uncompetitive.

39. COMPETITIVE VS NON COMPETITIVE

40. COMPETITIVE VS NON COMPETITIVE Competitive Examples – Enzyme- lactate dehydrogenase Substrate- lactate Inhibitor- Oxamate The enzyme of Krebs cycle succinate dehydrogenase catalyzes a reaction to convert succinate into fumarate. Malonate acts as a competitive inhibitor because it has a similar structure to succinate, the inhibitor binds to the active site of succinate dehydrogenase and inhibits the reaction. Noncompetitive Cyanide action on cytochrome oxidase is an example of non-competitive inhibition

41. Clinically Important Isoenzymes E N Z Y M E P R O F I L E I N L I V E R D I S E ASE Enzymes are commonly studied for diagnose liver diseases: Alanine aminotransferase (ALT) Aspartate aminotransferase (AST) Alkaline phosphatase (ALP) Nucleotide phosphatase (NTP) Gamma glutamyl transferase (GGT)

42. Enzyme inhibitor is a molecule that lowers the activity of an enzyme by binding with the active site of the enzyme. It reduces the amount of products. Enzyme inducer is a molecule that increases the activity of an enzyme either by binding to the enzyme or by increasing the synthesis of the enzyme. Enzyme repression refers to a decrease in enzyme after a stimulus. Allosteric enzymes are enzymes that have an additional binding site for effector molecules other than the active site.

43. Enzyme induction A process in which a molecule (e.g. a drug) induces (i.e. initiates or enhances) the expression of an enzyme. An enzyme inducer is a type of drug which binds to an enzyme and increases its metabolic activity. Regulated by exposure to drugs and environmental chemicals leading to increased rates of metabolism. The cholesterol pathway   provides examples of enzyme induction. A lack of cholesterol leads to an increased synthesis of HMG-CoA reductase, an example of enzyme induction .

44.  ENZYME REPRESSION  Enzyme repression is  the mode by which the synthesis of an enzyme is prevented by repressor( small protein molecule ) molecules . The cholesterol pathway   provides examples of enzyme induction and repression

45. ALLOSTERIC ENZYMES REGULATION Allosteric enzymes are enzymes that have an additional binding site for effector molecules other than the active site. The binding brings about conformational changes, thereby changing its catalytic properties. .

46. Therapeutic and diagnostic applications of enzymes and isoenzymes I sozymes are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. 

47. ENZYME APPLICATION IN THERAPEUTICS Certain enzymes can be used to treat several diseases, e.g.: Certain types of leukemia are treated with bacterial asparaginase. Dermal ulcers and severe burns can be cleaned with collagenas e by removing dead tissue. Penicillinase - Treats patients who have an allergy to penicillin. Streptokinase and urokinase - To dissolve a clot caused by myocardial infarctions. GIT disorders and chronic pancreatitis are treated with pepsin, lipase, amylase elastase, and trypsin peptidase.

48. Isoenzymes Isoenzymes or isozymes are multiple forms of same enzyme that catalyze the same chemical reaction Different chemical and physical properties: Kinetic properties Amino acid sequence Amino acid composition Multiple forms of the same enzyme will also help in the regulation of enzyme activity, Many of the isoenzymes are tissue-specific. Although isoenzymes of a given enzyme catalyse the same reaction, they differ in Km, Vmax or both. e.g.isoenzymes of LDH and CPK.

49. Clinically Important Isoenzymes L A C T A T E D E H YDRO G E N ASE It has five isoenzymes present in blood, ie., 1.LDH1 (HHHH) specific for heart--faster LDH2 (HHHM) RBCs--faster LDH3 (HHMM) brain--fast 4.LDH4 (HMMM) Liver--slow 5.LDH5 (MMMM) Muscle--slowest

50. Clinically Important Isoenzymes C R E AT I NE K I N ASE ( C K ) Three isoforms are present, i.e., 1.CK1 (BB) (brain) CK2 (MB) (heart) CK3 (MM) (skeletal muscle) In myocardial infarction (MI) there is elevation of total CK with marked elevation of CK2 (MB) after 4-8 hours. Electrophoresis is important to detect which fraction increased. CK2 is used for early detection. LDH1 for follow-up

51. ENZYME APPLICATION IN DIAGONSITIC Liver, cardiac and skeletal enzyme markers: Important markers that we are going to see are, Transaminase or aminotransferase. Alkaline phosphatase. Glutamate dehydrogenase (GLD). Transaminase or aminotransferase: Elevated levels of Aspartate aminotransferase (AST) and plasma alanine aminotransferase (ALT) are found  to indicate liver disease. As AST concentration in heart muscle is relatively high after myocardial infarction, AST level increases.

52. ISOENZYME APPLICATION Diagnosis of diseases: the measurement of serum alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) levels can help diagnose liver and bone diseases. Prognosis of diseases:  Isoenzymes can also be used to predict the outcome of a disease or the response to therapy. For example, the ratio of two isoenzymes of lactate dehydrogenase (LDH) can help predict the outcome of testicular cancer.

53. ISOENZYME APPLICATION Monitoring of therapy:   Isoenzymes can be used to monitor the response to therapy or the recurrence of a disease. For example, the measurement of serum thyroglobulin ( Tg ) levels can help monitor the recurrence of thyroid cancer. Targeted therapy:  Isoenzymes can be targeted by drugs to achieve selective effects on specific tissues or organs. For example, selective inhibitors of cyclooxygenase-2 (COX-2) isoenzyme are used to treat inflammation and pain

54. DEFINE & FUNCTION Define-Coenzymes are a type of cofactor and they are bound to enzyme’s active sites to aid with their proper functioning. The function of coenzymes is to transport groups between enzymes

55. Important coenzyme Alcohol dehydrogenase Coenzyme A Flavin adenine dinucleotide (FAD) Nicotinamide adenine dinucleotide (NAD) Adenosine triphosphate (ATP)

56. Adenosine triphosphate (ATP) The function of ATP is to transport chemical energy within cells for metabolism. ATP is often referred to as the energy currency of cells. Adenosine triphosphate is composed of an adenine nucleotide base, a ribose sugar and three phosphate groups. Energy can be released from ATP when the terminal phosphate group is released in a hydrolysis reaction.

57. END…..