UNIT 5 B.pharma 1st year 2nd sem .pptx ENZYME

This ppt is improve your knowledge regarding biochemistry enzyme

1. Bio-CHEMISTRY b. Pharma 2 nd semester unit-5 th Mr. Bulet Kumar Gupta Assistant Professor Sai College of Pharmacy, Mau

2. ENZYMES 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. OR Enzymes may be defined as biocatalysts synthesized by living cells. They are protein in nature (exception – RNA acting as ribozyme), colloidal and thermolabile in character, and specific in their action. In 1878, Kuhne used the word enzyme (Greek : in yeast) to indicate the catalysis taking place in the biological systems. The regulation of enzymes has been a key element in clinical diagnosis because of their role in maintaining life processes.

3. Chemical Nature & Properties Of Enzymes Enzymes are complex macromolecules with high molecular weight. They catalyze biochemical reactions in a cell. They help in the breakdown of large molecules into smaller molecules or bring together two smaller molecules to form a larger molecule. Enzymes do not start a reaction. However, they help in accelerating it. Enzymes affect the rate of biochemical reaction and not the direction. Enzymes are specific in action. Enzymatic activity decreases with increase in temperature. They show maximum activity at an optimum pH of 6 – 8. The velocity of enzyme increases with an increase in substrate concentration and then, ultimately reaches maximum velocity.

4. The functional unit of the enzyme is known as Holoenzyme which is often made up of Apoenzyme (the protein part) and a Coenzyme (non-protein organic part). Holoenzyme Apoenzyme + Coenzyme (active enzyme) (protein part) (non-protein part) The term prosthetic group is used when the non-protein moiety tightly (covalently) binds with the apoenzyme. The coenzyme can be separated by dialysis from the enzyme while the prosthetic group cannot be. The word monomeric enzyme is used if it is made up of a single polypeptide e.g. ribonuclease, trypsin.

5. NOMENCLATURE The International Union of Biochemistry and Molecular Biology is entrusted with designating names to enzymes in addition to assigning a number in order to identify them. Apart from a few originally studied enzymes such as rennin, pepsin and trypsin, almost all the enzyme names end in “ase”.  The nomenclature developed by the International Union of Biochemistry and Molecular Biology has something called EC numbers where each enzyme is preceded by EC. The first number in this series classifies this enzyme on the basis of its mechanism.

6. EC Numbers There are six groups of enzymes as per the reaction that is being catalyzed. Therefore, all enzymes are designated as “EC numbers”.  EC number is a 4 digit number for instance – a.b.c.d . Here “a” is class, “b” is subclass, “c” is sub-subclass and “d” is the sub-sub-subclass.  Example – EC number of Alcohol: NAD+ oxidoreductase is 1.1.1.1

7. ENZYMES CLASSIFICATION According to the International Union of Biochemists (I U B), enzymes are divided into six functional classes and are classified based on the type of reaction in which they are used to catalyze. The six kinds of enzymes are Hydrolases, Oxidoreductases, Lyases, Transferases, Ligases And Isomerases . EC 1 Oxidoreductases:  Oxidoreductases generally consist of a large class of enzymes, which catalyse the electronic transfer from reductant (an electron donor) to oxidant (an electron acceptor) molecule. Oxidoreductase also fulfills an essential role in both anaerobic mechanisms and aerobic metabolism.

8. EC 2 Transferases:  Transferases enzymes catalyze transfer amongst a group of atoms. These atoms are amine, carbonyl, carboxyl, acyl , methyl, phosphoryl and glycol form a donor substrate to the acceptor compound.  EC 3 Hydrolases:   Hydrolase’s enzymes catalyze the hydrolysis among the proteins, starch,fats, nucleic acid and various macromolecular substances.  EC 4 Lyases:  Lyases enzymes, catalyse the process of addition and removal of several elements of water (hydrogen and oxygen), CO2  (c arbon and oxygen) or Ammonia (nitrogen and hydrogen) into double bonds. 

9. EC 5 Isomerases:  Isomerases catalyse the isomerization changes within one singular molecule. Isomerases also involve a catalyzed reaction in a molecules’ structural rearrangement. For instance, isomerases catalyze “L- alanine’s conversion” into an isomeric form that is “D- alanine ”. EC 6 Ligases:  Ligases play important roles in maintaining genomic integrity by joining the breaks in DNA’s phosphodiester backbone.

10. FACTORS AFFECTING ENZYME ACTIVITY Concentration of Enzymes Concentration of Substrate Effect of temperature Effect of PH Effect of Product concentration Effect of Activator Effect of Time Effect of light & radiation

11. ACTIVE SITE Enzymes are big in size compared to substrates which are relatively smaller. Evidently, a small portion of the huge enzyme molecule is directly involved in the substrate binding and catalysis. The active site of an enzyme represents as the small region at which the substrate(s) binds and participates in the catalysis. Enzyme kinetics and Km value The enzyme (E) and substrate (S) combine with each other to form an unstable enzyme substrate complex (ES) for the formation of product. Here k1, k2 and k3 represent the velocity constants for the respective reactions

12. The first step of the equation, which is reversible, has the reaction rate constant of k +1  to produce the enzyme substrate complex and k -1  for the reverse reaction. The reaction rate constant for the second step of the equation, which is not reversible, is k +2 . The study of the rate at which an works is called enzyme kinetics. In a mathematical description of enzyme action developed by Leonor Michaelis and Maud Menten in 1913 , two constants, Vmax and Km. Michaelis-Menten model • Reaction involving invertase Degrades sucrose to glucose & fructose. Relation between reaction velocity and substrate concentration. . Hyperbola curve.

13. Vmax and Km • Vmax - the maximum rate of reaction when all enzyme active sites are saturated with substrate. Km- The substrate concentration that gives half maximal velocity. Km is a measure of the affinity an enzyme has for its substrate, as a lower Km means that less of the substrate is required to reach half of Vmax. Km (Michaelis constant) • Concentration of substrate at which reaction velocity reaches half of its maximum • Varies from enzyme to enzyme. Depends on temperature, nature of substrate, pH, ionic strength.

14. Km, the Michaelis-Menten constant (or Brig’s and Haldane’s constant), is given by the formula The following equation is obtained after suitable algebraic manipulation The following equation is obtained after suitable algebraic manipulation.

15. The Lineweaver-Burk plot Lineweaver-Burk double reciprocal plot : For the determination of Km value, the substrate saturation curve is not very accurate since Vmax is approached asymptotically. By taking the reciprocals of the equation (1), a straight line graphic representation is obtained. It is much easier to calculate the Km from the intercept on X-axis which is –(1/Km). Further, the double reciprocal plot is useful in understanding the effect of various inhibitions.

16. ENZYME INHIBITION Enzyme inhibitor is defined as a substance which binds with the enzyme and brings about a decrease in catalytic activity of that enzyme. The inhibitor may be organic or inorganic in nature. There are three broad categories of enzyme inhibition Reversible inhibition. Irreversible inhibition. Allosteric inhibition. 1- Reversible inhibition- The inhibitor binds non-covalently with enzyme and the enzyme inhibition can be reversed if the inhibitor is removed. The reversible inhibition is further sub-divided into------ A). Competitive inhibition B). Non-competitive inhibition

17. Competitive Inhibition Competitive inhibition involves a molecule, other than the substrate, binding to the enzyme’s  active site The molecule (inhibitor) is structurally and chemically similar to the substrate (hence able to bind to the active site) The competitive inhibitor blocks the active site and thus prevents substrate binding

18. Selected examples of enzymes with their respective substrates and competitive inhibitors ENZYME SUBSTRATE INHIBITORS SIGNIFICANCE OF INHIBITORS Xanthine oxidase Hypoxanthine Allopurinol Used in the control of gout to reduce excess production of uric acid from hypoxanthine. Monoamine oxidase Catecholamines Ephedrine Amphetamine Useful for elevating catecholamine levels. Dihydrofolate reductase Dihydrofolic acid Aminopterin, AmethopterinMethotrexate Employed in the treatment of leukemia and other cancers. Acetylcholine esterase ACH Succinylcholine Used in surgery for muscle relaxation, in anaesthetised patients. Dihydropteroate synthase P-aminobenzoic acid (PABA) Sulfonilamide Prevents bacterial synthesis of folic acid.

19. Non-competitive Inhibition Non-competitive inhibition involves a molecule binding to a site other than the active site (an  allosteric site ) The binding of the inhibitor to the allosteric site causes a conformational change to the enzyme’s active site As a result of this change, the active site and substrate no longer share specificity, meaning the substrate cannot bind As the inhibitor is  not  in direct competition with the substrate, increasing substrate levels cannot mitigate the inhibitor’s effect

20. 2. Irreversible inhibition(Poisons)- The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. These inhibitors are usually toxic substances that poison enzymes. Iodoacetate is an irreversible inhibitor of the enzymes like papain and glyceraldehyde 3-phosphate dehydrogenase. Iodoacetate combines with sulfhydryl (-SH) groups at the active site of these enzymes and makes them inactive. 3. Allosteric inhibition- The details of this type of inhibition are given under allosteric regulation as a part of the regulation of enzyme activity in the living system.

21. Enzyme inhibition by drugs Enzymes are the natural targets for development of pharmacologic agents. Many of the drugs used in the treatment of diseases act as enzyme inhibitors. For example : 1) Cholesterol lowering statins drugs (lovastatin) inhibit the enzyme HMG CoA reductase. 2) Drugs (tenofovir, emtricitabine) employed to block HIV replication inhibit the enzyme viral reverse transcriptase. 3) Hypertension is often treated by the drugs (captopril, enalapril )which inhibit angiotensin converting enzyme.

22. REGULATION OF ENZYME IN LIVING SYSTEMS In biological system, regulation of enzyme activities occurs at different stages in one or more of the following ways to achieve cellular economy. 1. Allosteric regulation 2. Activation of latent enzymes 3. Compartmentation of metabolic pathways 4. Control of enzyme synthesis 5. Enzyme degradation 6. Isoenzymes

23. Allosteric Enzyme regulation and allosteric inhibition Some of the enzymes possess additional sites, known as allosteric sites (Greek : allo–other), besides the active site. Such enzymes are known as allosteric enzymes. The allosteric sites are unique places on the enzyme molecule. Allosteric Inhibitors Allosteric Activators

24. Allosteric effectors: Certain substances referred to as allosteric modulators (effectors or modifiers) bind at the allosteric site and regulate the enzyme activity. The enzyme activity is increased when a positive (+) allosteric effector binds at the allosteric site known as activator site. On the other hand, a negative (–) allosteric effector binds at the allosteric site called inhibitor site and inhibits the enzyme activity. Enzymes that are regulated by allosteric mechanism are referred to as allosteric enzymes.

25. Enzyme Induction and Repression The term induction is used to represent increased synthesis of enzyme while repression indicates its decreased synthesis. Induction or repression which ultimately determines the enzyme concentration at the gene level through the mediation of hormones or other substances. Examples of enzyme induction: The hormone insulin induces the synthesis of glycogen synthetase, glucokinase, phosphofructokinase and pyruvate kinase. All these enzymes are involved in the utilization of glucose. The hormone cortisol induces the synthesis of many enzymes e.g. pyruvate carboxylase, tryptophan oxygenase and tyrosine aminotransferase.

26. Examples of repression: In many instances, substrate can repress the synthesis of enzyme. Pyruvate carboxylase is a key enzyme in the synthesis of glucose from non-carbohydrate sources like pyruvate and amino acids. If there is sufficient glucose available, there is no necessity for its synthesis. This is achieved through repression of pyruvate carboxylase by glucose.

27. ISOENZYME The multiple forms of an enzyme catalyzing the same reaction are isoenzymes or isozymes. They, however, differ in their physical and chemical properties which include the structure, electrophoretic and immunological properties, Km and Vmax values, pH optimum, relative susceptibility to inhibitors and degree of denaturation. EXISTENCE OF ISOENZYMES Many possible reasons are offered to explain the presence of isoenzymes in the living systems. 1. Isoenzymes synthesized from different genes e.g. malate dehydrogenase of cytosol is different from that found in mitochondria.

28. 2. Oligomeric enzymes consisting of more than one type of subunits e.g. lactate dehydrogenase and creatine phosphokinase. 3. An enzyme may be active as monomer or oligomer e.g. glutamate dehydrogenase. 4. In glycoprotein enzymes, differences in carbohydrate content may be responsible for isoenzymes e.g. alkaline phosphatase. Isoenzymes of lactate dehydrogenase (LDH) Among the isoenzymes, LDH has been the most thoroughly investigated. LDH whose systematic name is L-lactate NAD+ oxidoreductase catalyses the interconversion of lactate and pyruvate as shown below

29. LDH has five distinct isoenzymes LDH1, LDH2, LDH3, LDH4 and LDH5. They can be separated by electrophoresis (cellulose or starch gel or agarose gel). LDH1 has more positive charge and fastest in electrophoretic mobility while LDH5 is the slowest. Structure of LDH isoenzymes LDH is an oligomeric (tetrameric) enzyme made up of four polypeptide subunits. Two types of subunits namely M (for muscle) and H (for heart) are produced by different genes. M–subunit is basic while H subunit is acidic. The isoenzymes contain either one or both the subunits giving LDH1 to LDH5. The characteristic features of LDH isoenzymes are given in table-

30. Diagnostic importance of LDH Isoenzymes of LDH have immense value in the diagnosis of heart and liver related disorders.

31. Isoenzymes of creatine phosphokinase Creatine kinase (CK) or creatine phosphokinase (CPK) catalyses the inter-conversion of phosphocreatine (or creatine phosphate) to creatine. Creatine CPK ADP ATP Phosphocreatine CPK exists as three isoenzymes. Each isoenzyme is a dimer composed of two subunits—M (muscle) or B (brain) or both.

32. Liver, cardiac and skeletal enzyme markers A) Transaminase or aminotransferase: Elevated levels of Aspartate aminotransferase (AST) and plasma alanine aminotransferase (ALT) are found  to indicate liver disease. Alcohol consumption or the consumption of various drugs like salicylates, ampicillin, etc. are associated with moderate increases in AST and ALT. B) Alkaline phosphatase: Elevated ALP levels in the plasma are particularly useful in the diagnosis of bone and liver conditions.

33. C) Glutamate dehydrogenase (GLD): GLD is increased in the serum of patients with hepatocellular damage, offering differential diagnostic potential in the investigation of liver disease.  GLD is released from necrotic cells and is of value in estimation of the severity of liver cell damage. 2 ) Biliary tract enzymes: A) Alpha amylase -  Amylase measurements in the serum and urine are typically used to diagnose pancreatic diseases like pancreatitis, cholecystitis, or tumors.

34. B) Trypsin -  During the first six weeks after birth, serum trypsin levels can be measured to screen for cystic fibrosis.  Pancreatic ductulus are also screened with serum trypsin. C) Chymotrypsin -  A serum chymotrypsin level exceeding eight times the normal value indicates renal failure, as with amylase and trypsin.

35. The functional enzyme is referred to as holoenzyme which is made up of a protein part (apoenzyme) and a non-protein part (coenzyme). Coenzymes participate in various reactions involving transfer of atoms or groups like hydrogen, aldehyde, keto, amino, acyl, methyl, carbon dioxide etc. Coenzymes play a decisive role in enzyme function