Enzymes Biochemistry for Allied Health.pptx

An introduction to Enzymes for Health Science students

1. Department of Molecular Medicine KSMD, KNUST, Kumasi

2. Course outline Introduction to enzymes Characteristics of enzymes Name/Structure/Classes of enzymes Enzyme specificity Functions of enzymes 2 C. Obirikorang

3. Course outline Mechanism of enzymes/ Models for enzyme action Cofactor/ Coenzymes/Prosthetic Groups Factors affecting reaction rate Enzyme Inhibition Enzyme Regulation Application of Enzymes 3 C. Obirikorang

4. Enzymes 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. Most enzymes have tertiary and quaternary structures Catalysts for biological reactions. Teacher-Student relationship is a good example. 4 C. Obirikorang

5. Enzymes Remains unchanged in overall process. Reactants bind to enzymes, products are released. Activity lost if denatured. May contain cofactors such as metal ions or organic (vitamins) Does not affect the equilibrium of the reaction. 5 C. Obirikorang

6. Characteristics of Enzymes Accelerate the rate of making and breaking of covalent catalysis. Highly specific: React with one substrate Not changed in the reaction. Catalyze many cycles (turnovers) of the reaction. 6 C. Obirikorang

7. Characteristics of Enzymes Usually proteins, but can be RNA. Bind substrates in special regions called ‘active sites’. Catalyze the forward and reverse reactions equal extent. They do not change the position of the equilibrium. Function by stabilizing the transition state. 7 C. Obirikorang

8. Nomenclature of Enzymes In the early days, the enzymes were given names by their discoverers in an arbitrary manner. Names like pepsin, trypsin and chymotrypsin convey no information about the function of the enzyme or the nature of the substrate on which they act. Sometimes, the suffix- ase was added to the substrate for naming the enzymes e.g. lipase acts on lipids; nuclease on nucleic acids; lactase on lactose. These are known as trivial names and offered no complete information about the enzyme.

9. Enzymes are sometimes considered under two broad categories : Intracellular enzymes – They are functional within cells where they are synthesized. Extracellular enzymes – These enzymes are active outside the cell; all the digestive enzymes belong to this group. Nomenclature of Enzymes

10. The International Union of Biochemistry (IUB)appointed an Enzyme Commission in 1961. This committee devised some basic principles for the classification and nomenclature of enzymes. 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 Nomenclature of Enzymes

11. Oxidoreductases (EC 1) : Enzymes involved in oxidation-reduction reactions. Transferases (EC 2) : Enzymes that catalyse the transfer of functional groups. Hydrolases (EC 3) : Enzymes that bring about hydrolysis of various compounds. Classes of enzymes

12. Lyases (EC 4) : Enzymes specialised in the addition or removal of water, ammonia, CO2 etc. Isomerases (EC 5) : Enzymes involved in all the isomerization reactions. Ligases (EC 6) : Enzymes catalysing the synthetic reactions (Greek : ligate—to bind) where two molecules are joined together and ATP is used. Classes of enzymes

13. Structure of enzymes are in general globular proteins and range from just 62 amino acid residues in size to over 2,500 residues. Most enzymes are much larger than the substrates they act on. only a small portion of the enzyme (around 2–4 amino acids) is directly involved in catalysis. The region that contains these catalytic residues, binds the substrate, and then carries out the reaction is known as the active site. 14 C. Obirikorang

14. Structure of enzymes Enzymes are long, linear chains of amino acids that fold to produce a three-dimensional product. Each unique amino acid sequence produces a specific structure, which has unique properties.  Most enzymes can be denatured—that is, unfolded and inactivated—by heating or chemical denaturants, which disrupt the three-dimensional structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible. 15 C. Obirikorang

15. Active Site Enzymes are big in size compared to substrates which are relatively smaller. 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.

16. Features of the Active Site The existence of active site is due to the tertiary structure of protein resulting in three dimensional native conformation. The active site is made up of amino acids ( catalytic residues ) which are far from each other in the linear sequence of amino acids. For instance, the enzyme lysozyme has 129 amino acids. The active site is formed by the contribution of amino acid residues numbered 35, 52, 62, 63 and 101.

17. Active sites are regarded as clefts or crevices or pockets occupying a small region in a big enzyme molecule. The active site is not rigid in structure and shape. It is rather flexible to promote the specific substrate binding. Generally, the active site possesses a substrate binding site and a catalytic site. The latter is for the catalysis of the specific reaction. Features of the Active Site

18. The coenzymes or cofactors on which some enzymes depend are present as a part of the catalytic site. The substrate(s) binds at the active site by weak noncovalent bonds . Enzymes are specific in their function due to the existence of active sites. Features of the Active Site

19. The commonly found amino acids at the active sites are serine, aspartate, histidine, cysteine, lysine, arginine, glutamate, tyrosine etc. Among these amino acids, serine is the most frequently found. The substrate[S] binds the enzyme (E) at the active site to form enzyme-substrate complex (ES). The product (P) is released after the catalysis and the enzyme is available for reuse. Features of the Active Site

20. Enzyme Specificity are usually very specific as to which reactions they catalyze and the substrates that are involved in these reactions.  Complementary shape, charge and hydrophilic/hydrophobic characteristics of enzymes and substrates are responsible. Enzymes can also show impressive levels of  stereospecificity regioselectivity  and   chemoselectivity .  21 C. Obirikorang

21. Enzyme Specificity Some enzymes do ‘proof reading’ after each step. An enzyme such as DNA polymerase  catalyzes a reaction in a first step and then checks that the product is correct in a second step. This two-step process results in average error rates of less than 1 error in 100 million reactions  22 C. Obirikorang

22. BIOLOGICAL FUNCTION OF ENZYMES They are indispensable for signal transduction and cell regulation, often via kinases and phosphatases . An important function of enzymes is in the digestive systems of animals. Several enzymes can work together in a specific order, creating metabolic pathways. Enzymes are also involved in some exotic functions e.g. Luciferase generating light in fireflies 23 C. Obirikorang

23. MECHANISM OF ENZYME CATALYSIS Enzyme catalysis occurs after the substrate S binds to the enzyme E to form an enzyme-substrate complex ES. This ES complex then undergoes catalytic alteration to form the product P and reform the (uncomplexed) enzyme E. The enzyme E is unchanged in the process and is free to participate in another round of catalysis 24 C. Obirikorang

24. Enzyme Mechanism Enzymes can act in several ways, all of which lowers the activation energy: Creating an environment in which the transition state is stabilized. Lowering the energy of the transition state Providing an alternative pathway.  Reducing the reaction entropy change by bringing substrates together in the correct orientation to react.  Increases in temperatures speed up reactions. 25 C. Obirikorang

25. MECHANISM OF ENZYME ACTIVITY The energy diagram shows that an enzyme lowers the activation energy of a reaction. This is how an enzyme can increase the rate of reaction. A lower energy barrier means substrate is converted into product at a faster rate. 26 C. Obirikorang

26. Catalytic Power Enzymes can accelerate reactions as much as 10 16 over uncatalyzed rates! Urease is a good example: Catalyzed rate: 3x10 4 /sec Uncatalyzed rate: 3x10 -10 /sec Ratio is 1x10 14 ! 27 C. Obirikorang

27. Lock and key model This theory was proposed by a German biochemist, Emil Fischer in 1894. This is the very first model proposed to explain an enzyme catalyzed reaction . According to this model, the structure or conformation of the enzyme is rigid . The substrate fits to the binding site (now active site) just as a key fits into the proper lock. Thus the active site of an enzyme is a rigid and pre-shaped template where only a specific substrate can bind. 28 C. Obirikorang

28. This model does not give any scope for the flexible nature of enzymes. The model therefore totally fails to explain many facts of enzymatic reactions . The most important being the effect of allosteric modulators . Lock and key model

29. Induced Fit Model In 1958, Daniel Koshland suggested a modification to the lock and key model. The induced fit model. As per his model, the active site is NOT rigid and pre-shaped. Enzymes are rather flexible structures, the active site is continuously reshaped by interactions with the substrate as the substrate interacts with the enzyme. This model was therefore able to explain the action of allosteric modulators and competitive inhibition on enzymes 30 C. Obirikorang

30. INDUCED FIT MODEL The induced fit hypothesis states that the shape of the active site changes in the presence of the substrate to yield a precise fit. 31 C. Obirikorang

31. Substrate Strain Theory In this model, the substrate is strained due to the induced conformation change in the enzyme. It is also possible that when a substrate binds to the preformed active site, the enzyme induces a strain to the substrate . The strained substrate leads to the formation of product. A combination of the induced fit model with the substrate strain is considered to be operative in the enzymatic action.

32. Substrate Strain Theory

33. Cofactor Some enzymes do not need any additional components to show full activity. However, others require non-protein molecules called cofactors to be bound for activity is a non-protein chemical compound that is bound to an enzyme.  Cofactors can be considered "helper molecules" that assist in biochemical transformations. 34 C. Obirikorang

34. Cofactor Are either organic or inorganic. classified depending on how tightly they bind to an enzyme. Loosely bound- Coenzyme Tightly bound- Prosthetic group. 35 C. Obirikorang

35. PROSTHETIC GROUPS Prosthetic groups are distinguished by their tight, stable incorporation into a protein’s structure by covalent or noncovalent forces e.g. pyridoxal phosphate, flavin mononucleotide (FMN), flavin dinucleotide (FAD), thiamin pyrophosphate, biotin, and the metal ions of Co, Cu, Mg, Mn , Se, and Zn ( metalloenzymes ). 36 C. Obirikorang

36. Coenzyme Coenzymes are small organic molecules that can be loosely bound to an enzyme.  Coenzymes transport chemical groups from one enzyme to another. Organic co-enzymes – Thiamin, Riboflavin, Niacin, Biotin Inorganic co-enzymes – Mg ++ , Fe ++ , Zn ++ , Mn ++ Enzyme + Co-enzyme = holoenzyme Enzyme alone = apoenzyme 37 C. Obirikorang

37. FACTORS AFFECTING ENZYME ACTIVITY 38 C. Obirikorang

38. Temperature Enzymes typically operate best in a relatively narrow range of environmental conditions. Like all proteins, enzymes are made as long, linear chains of amino acids that fold to give a 3-D product. As temp. rises, Enzyme activity increases because there are more molecular collisions. 39 C. Obirikorang

39. Effect of Temperature The temperature has a significant effect on enzyme activity. Most enzymes function well within a certain temperature range, and they become less active or irreversibly denatured if the temperature is raised above that range.

40. Temperature Enzyme activity declines rapidly when enzyme is denatured at a certain T-that is, unfolded and inactivated by heating, which destroys the 3-D structure of the protein. Depending on the enzyme, denaturation may be reversible or irreversible resulting in change in shape of enzyme. 41 C. Obirikorang

41. Temperature Clinical significance : Foods can be preserved in refrigerators (at low temperatures) due to reduced bacterial enzyme activities. Certain surgeries are carried out by lowering the patient’s body temperature ( induced hyporthermia ), and thus the metabolic rate.

42. EFFECT OF TEMPERATURE 43 C. Obirikorang

43. EFFECT OF pH Increase in the hydrogen ion concentration (pH) considerably influences the enzyme activity and a bell-shaped curve is normally obtained Each enzyme has an optimum 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. Hydrogen ions influence the enzyme activity by altering the ionic charges on the amino acids ( particularly at the active site ), substrate, ES complex

44. EFFECT OF pH 45 C. Obirikorang

45. Substrate Concentration To achieve max product per unit time, enough substrate is needed to fill active sites. At lower concentrations, the active sites on most of the Enzyme molecules are not filled because there is not much Substrate. Higher concentration of substrate cause more collisions between the molecules. 47 C. Obirikorang

46. Substrate Concentration With more molecules and collisions, Enzymes are more likely to encounter molecules of substrate. The maximum velocity of a reaction is reached when the active sites are almost continuously filled. Increased Substrate concentration after this point will not increase the rate. Reaction rate therefore increases as substrate concentration is increased but it levels off. 48 C. Obirikorang

47. EFFECT OF SUBSTRATE CONCENTRATION 49 C. Obirikorang

48. As the concentration of the enzyme is increased, the velocity of the reaction proportionately increases. This property of enzyme is made use in determining the serum enzymes for the diagnosis of diseases. By using a known volume of serum, and keeping all the other factors (substrate, pH, temperature etc.) at the optimum level, the enzyme could be assayed in the laboratory. ENZYME CONCENTRATION

49. ENZYME CONCENTRATION If insufficient Enzyme is present, the reaction will not proceed as fast as it otherwise would because there is not enough Enzyme for all of the reactant molecules. As the amount of E is increased, the rate of reaction increases. If there are more enzyme molecules than are needed, adding additional enzyme will increase the rate. 51 C. Obirikorang

50. ENZYME CONCENTRATION

51. Effects 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. In the living system, this type of inhibition is generally prevented by a quick removal of products formed. The end product inhibition by feedback mechanism

52. Effect of Light and Radiation Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain enzymes due to the formation of peroxides. UV rays inhibit salivary amylase activity.

53. Effects of Activators Some of the enzymes require certain inorganic metallic cations like Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, K+ etc. for their optimum activity Metals function as activators of enzyme velocity through various mechanisms— These include combining with the substrate, formation of ES-metal complex, direct participation in the reaction and bringing a conformational change in the enzyme.

54. Enzyme Inhibitors C Obirikorang 56

55. 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 two broad categories of enzyme inhibition Reversible inhibition. Irreversible inhibition.

56. ENZYME INHIBITORS Inhibitors: compounds that decrease the activity of enzymes Most drugs are enzyme inhibitors. Inhibitors are also important for determining enzyme mechanisms and nature of the active site. Important to know how inhibitors work facilitates drug design, inhibitor design 58 C Obirikorang

57. ENZYME INHIBITION Inhibitor – substance that binds to an enzyme and interferes with its activity Can prevent formation of ES complex or prevent ES breakdown to E + P. Irreversible and Reversible Inhibitors Irreversible inhibitor binds to enzyme through covalent bonds (binds irreversibly), slow dissociation Reversible Inhibitors bind through non-covalent interactions (disassociates from enzyme easily) 59 C Obirikorang

58. TYPES OF ENZYME INHIBITION Reversible inhibition (inhibitors that can reversibly bind and dissociate from enzyme, activity of enzyme recovers when inhibitor is diluted out, usually in a non-covalent interaction) Competitive Mixed (noncompetitive) Uncompetitive Irreversible inhibition (Inactivators that irreversibly associate with enzyme, activity of enzyme does not recover with dilution or removal of inhibitor, usually by covalent interaction) 60 C Obirikorang

59. 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 sub-divided into Competitive inhibition Non-competitive inhibition Uncompetitive inhibition

60. Competitive Inhibition The inhibitor (I) which closely resembles the real substrate (S) is regarded as a substrate analogue . The inhibitor competes with substrate and binds at the active site of the enzyme but does not undergo any catalysis. As long as the competitive inhibitor holds the active site, the enzyme is not available for the substrate to bind. During the reaction, ES and EI complexes are formed

61. The relative concentration of the substrate and inhibitor and their respective affinity with the enzyme determines the degree of competitive inhibition. The inhibition could be overcome by a high substrate concentration. Competitive Inhibition

62. Competitive Inhibition

63. Examples of Competitive Inhibition in Medicine

64. Non Competitive Inhibition The inhibitor binds at a site other than the active site on the enzyme surface (allosteric site). This binding impairs the enzyme function. The inhibitor has no structural resemblance with the substrate The inhibitor can bind to the free enzyme or the enzyme-substrate complex

65. Non Competitive Inhibition

66. Uncompetitive Inhibition Also known as anti-competitive inhibition. Occurs when the enzyme inhibitor binds to ONLY the enzyme-substrate complex. The inhibitor usually binds to an allosteric site and causes conformational changes in the enzyme.

67. Uncompetitive Inhibition

68. Irreversible Inhibition The inhibitors bind covalently with the enzymes and inactivate them, which is irreversible. These inhibitors are usually toxic substances that poison enzymes.

69. Examples Irreversible Inhibition Diisopropyl fluorophosphate (DFP) is a nerve gas developed by the Germans during Second World War. DFP irreversibly binds with enzymes containing serine at the active site, e.g. serine proteases , acetylcholinesterase . Leads to constriction of the pupils, profuse salivation, convulsion and involuntary urination and defecation. Death is usually by cardiac arrest due to lost of respiratory functions

70. Many organophosphorus insecticides are toxic to animals (including man) They block the activity of acetylcholine esterase (essential for nerve conduction), resulting in paralysis of vital body functions. Antibiotics act as irreversible inhibitors of serine – containing enzymes, and block the bacterial cell wall synthesis. Disulfiram (Antabuse®) is a drug used in the treatment of alcoholism . It irreversibly inhibits the enzyme acetaldehyde dehydrogenase leading to the accumulation of acetaldehyde (responsible for hangover). Examples Irreversible Inhibition

71. Enzyme Regulation 73 C Obirikorang

72. Enzyme Regulation in Living Systems Regulation of enzyme activities occurs at different stages in one or more of the following ways to achieve cellular economy. Allosteric regulation Activation of latent enzymes Compartmentation of metabolic pathways Control of enzyme synthesis Enzyme degradation lsoenzymes

73. Allosteric Regulation Some of the enzymes possess additional sites, known as allosteric site besides the active site. Such enzymes are known as allosteric enzymes. The allosteric sites are unique places on the enzyme molecule

74. Allosteric Regulation 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

75. Feedback Regulation/Inhibition The process of inhibiting the first step by the final product , in a series of enzyme catalysed reactions of a metabolic pathway is referred to as feedback regulation/inhibition . This is a real cellular economy to save the cell from the wasteful expenditure of synthesizing a compound which is already available within the cell.

76. Feedback Regulation/Inhibition Feedback inhibition or end product inhibition is a specialized type of allosteric inhibition necessary to control metabolic pathways for efficient cellular function

77. Latent Enzymes Some enzymes are synthesized as Proenzymes or zymogens which undergo irreversible covalent activation by the breakdown of one or more peptide bonds. For instance, proenzymes -namely chymotrypsinogen , pepsinogen and plasminogen, are respectively - converted to the active enzymes chymotrypsin, pepsin and plasmin

78. Latent Enzymes Certain enzymes exist in the active and inactive forms which are interconvertible, depending on the needs of the body. The interconversion is brought about by the reversible covalent modifications. Namely phosphorylation and dephosphorylation , and oxidation and reduction of disulfide bonds.

79. Glycogen Phosphorylase Glycogen phosphorylase is a muscle enzyme that breaks down glycogen to provide energy. This enzyme is a homodimer (two identical subunits) and exists in two interconvertible forms. Phosphorylase b ( dephospho enzyme) is inactive. Converted by phosphorylation of serine residues to active form phosphorylase a.

80. Compartmentalization There are certain substances in the body (e.g., fatty acids, glycogen) which are synthesized and also degraded. There is no point for simultaneous occurrence of both the pathways. Generally, the synthetic (anabolic) and breakdown (catabolic) pathways are operative in different cellular organelles to achieve maximum economy

81. Compartmentalization For instance, enzymes for fatty acid synthesis are found in the cytosol Whereas enzymes for fatty acid oxidation are present in the mitochondria . Depending on the needs of the body - through the mediation of hormonal and other controls -fatty acids are either synthesized or oxidized

82. Enzyme Synthesis Most of the enzymes, (particularly the rate limiting ones) are present in very low concentration. Nevertheless, the amount of the enzyme directly controls the velocity of the reaction, catalysed by that enzyme. Many rate Iimiting enzymes have short half-lives. This helps in the efficient regulation of the enzyme level

83. Enzyme Synthesis There are two types of enzymes- Constitutive enzymes (house-keeping enzymes)-The levels of which are not controlled and remain fairly constant. Inducible enzymes - Their concentrations increase or decrease as per body needs and are well-regulated. The synthesis of enzymes (proteins) is regulated by the gene

84. Induction and Repression Induction is used to represent increased synthesis of enzyme while repression indicates its decreased synthesis . Induction or repression determines the enzyme concentration at the gene level. Induction or repression is mediated by hormones or other substances

85. Induction and Repression The hormone insulin induces the synthesis of glycogen synthetase , glucokinase , phosphofructokinase and pyruvate kinase . All these enzymes are used in the utilization of glucose Substrate can repress the synthesis of enzyme. lf there is sufficient glucose available, there is no necessity for its synthesis. (Pyruvate carboxylase)

86. Enzyme Degradation Enzymes are not immortal. There is a lot of variability in the half-lives of individual enzymes. For some, it is in days while for others in hours or in minutes, LDH- 5 to 6 days amylase -3 to 5 hours.

87. Unit of Enzyme Activity Enzymes are never expressed in terms of their concentration (as mg or pg etc.), Rather expressed only as activities . Various methods have been introduced for the estimation of enzyme activities (particularly for the plasma enzymes). In fact, the activities have been expressed in many ways, like King-Armstrong units , Somogyi units , Reitman-Frankel units , spectrophotometric units etc

88. Katal The need to maintain uniformity in the expression of enzyme activities. A new unit- namely katal (abbreviated as kat )-was introduced by EC. One kat denotes the conversion of one mole substrate per second ( mol /sec). Activity may also be expressed as millikatals ( mkat ), microkatals ( pkat ) and so on.

89. International Unit (IU) Some workers prefer to use standard units or Sl units (System International). One International Unit ( lU ) is defined as the amount of enzyme activity that catalyzes the conversion of one micromole of substrate per minute. 1 UI = 60 μ katal

90. Non-Protein Enzymes (Ribozymes) Ribozymes are a group of ribonucleic acids that function as biological catalysts They are therefore regarded as non-protein enzyme Altman and his coworkers, in 1983, found ribonuclease P

91. Non-Protein Enzymes (Ribozymes) The RNA part isolated from ribonuclease P exhibited a true enzyme activity and also obeyed Michaelis-Menten kinetics. Later studies have proved that RNA, in fact, can function as an enzyme and bring about the catalysis. RNA molecules are known to adapt a tertiary structure just as in the case of enzymes.

92. Application of Enzymes

93. Therapeutic Application Enzyme Application Streptokinase/ urokinase To remove blood clots   Asparaginase   In cancer therapy   Papain   Anti-inflammatory   α-Antitrypsin To treat emphysema (breathing difficulty due to distension of lung

94. Analytical Application Enzyme Application Urease Urea   Cholesterol oxidase   Cholesterol   Uricase   Uric acid   Lipase Triacylglycerols Luciferase To detect bacterial contamination of foods Horse Radish peroxidase Used in ELISA

95. Application in Genetics Enzyme Application Restriction endonucleases   Gene transfer, DNA finger printing DNA polymerase Polymerase chain reaction

96. Industrial Application Enzyme Application Rennin   Cheese preparation   Glucose isomerase Production of high fructose syrup α-Amylase   In food industry to convert starch to glucose Proteases Washing powder

97. Enzymes in Disease Diagnosis Enzyme Disease condition Amylase   Acute pancreatitis   Alanine transaminase (ALT)   Liver diseases (hepatitis)   Aspartate transaminase (AST)   Heart attacks (myocardial infarction)   Alkaline phosphatase   Rickets, obstructive jaundice   Acid phosphatase   Cancer of prostate gland  

98. Enzymes in Disease Diagnosis Lactate dehydrogenase (LDH) Heart attacks, liver diseases   Creatine phosphokinase (CPK)   Myocardial infarction (early marker)   Aldolase   Muscular dystrophy 5'-Nucleolidase   Hepatitis   Α-Glutamyl transaminase (GGT)   Alcoholism

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