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7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
7.27.10 enzymes   coloso
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7.27.10 enzymes coloso

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Lecture: Enzymes01

Lecture: Enzymes01

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  • 1. Enzymes: Mechanisms, Kinetics, and Regulation Relicardo M. Coloso, Ph. D. College of Medicine Central Philippine University
  • 2. What are enzymes? Most biological enzymes are proteins . They perform the chemical reactions in cells. Not all proteins are enzymes, but most enzymes are proteins (the exception is catalytic RNA). A catalyst is a molecule which increases the rate of a reaction but is not the substrate or product of that reaction. A substrate (A) is a molecule upon which an enzyme acts to yield a product (B). A ------> B Enzyme
  • 3. Enzyme – protein Primary structure; Secondary structure; Tertiary structure Substrate – in the active site of the enzyme
  • 4. Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes . Catalysis of biochemical reactions in the cell is vital due to the very low reaction rates of the uncatalysed reactions.
  • 5. Why study enzymes? Enzymes govern nearly all reactions that happen inside the cell. Virtually all reactions in cells are regulated by the activity of the enzyme that catalyzes the reaction. By studying enzyme activity we also learn the function of the chemical reaction in the cell .
  • 6. What does an enzyme do? Enzymes Catalyze Reactions Enzymes have affinity for the substrate in a transition state. They get the substrate into the right conformation which will lead to the breakdown into products. Alternatively, for a reaction such as the one shown below, the enzyme may increase the local concentration of the two substrates A and B, driving the reaction forward: A + B ---> C The part of the enzyme that does the work is called the active site . The residues in this site are in the right 3D conformation to accomplish the enzyme's work.
  • 7. The free energy of this reaction is not changed by the presence of the enzyme, but, for a favored reaction (where  G is negative), the enzyme can speed it up. Graph of the free energy against the reaction progress:  G* is the activation energy  G is negative overall for forward reaction
  • 8. Induced fit mechanism Substrate entering the active site of enzyme Products leaving active site of enzyme
  • 9. Diagrams shows the induced fit hypothesis of enzyme action. How does an enzyme catalyze a reaction? The favored model for the enzyme- substrate interaction is the induced fit model. This model proposes that the initial interaction between enzyme and substrate is relatively weak, but then leads to conformational changes in the enzymes that result in stronger interactions.
  • 10. Nomenclature: How do we name enzymes? Enzymes are named in a variety of ways. General rules of enzyme nomenclature: 1)Named for its substrate: substrate + -ase ex. lactase catalyzes lactose  glucose + galactose protease : protein  peptides + amino acids lipase : lipids  monoglycerides + fatty acids carbohydrase : carbohydrate  monosaccharides If you are lactose intolerant, you can buy lactase in a powdered form to help you digest the food. (An enzyme with this function, produced by the bacterium E. coli is called  -galactosidase.) 2)Named for its action ex. Deoxyribonuclease, or DNase catalyzes DNA ---> dNMP nucleotides the enzyme might be an endonuclease or an exonuclease
  • 11. How does one study enzyme activity? To study an enzyme , an assay is necessary. The assay is a measurement of a chemical reaction, which might involve measuring the (1)formation of the product . For example,  galactosidase catalyzes the following reaction:  -galactosidase (lactase) lactose ------------------------------> glucose + galactose For this reaction, measuring the formation of glucose would constitute an assay. Because this is technically difficult, an easier way to follow the reaction of glucose with another enzyme glucose oxidase + reagent o-dianisidine leading to the formation of a colored product which can be measured in a spectrophotometer (Abs 436 nm) .
  • 12. Another lactase assay method uses this reaction: The synthetic substrate used in this assay is  o-nitrophenyl--D galactoside (ONPG), which, upon hydrolysis of the  -galactosidic bond, yields galactose and  o-nitrophenol , a yellow compound (absorption max = 450 nm). Enzyme activity is proportional to the increase in A 450 during incubation.
  • 13. Steps in doing an enzyme assay: Add reaction mix with a pipettor Add the enzyme Mix and incubate for specified time Pour mixture into a cuvette Read absorbance in a spectrophotometer
  • 14. 2) Disappearance of the substrate For example,  -amylase catalyses the following reaction Starch maltose units Starch reacts with iodine ( yellow ) to form a blue complex (Abs 620 nm) Enzyme (amylase) will be incubated with starch. After incubation period, an aliquot of the mixture is combined with iodine. Acid stops the reaction and the iodine reacts with remaining starch to form blue color. Any remaining starch will turn blue, the color is proportional to the amount of starch. enzyme activity, amount of starch degraded, amount of starch reacting with iodine, absorbance at 620 nm.
  • 15. Types of enzyme mechanisms:
    • proton donor, acceptor; acid-base catalysis
    • Example: serine protease
  • 16. 2) ionic catalysis Example: carboxypeptidase (Zn +2 is a metal co-factor)
  • 17. 3) covalent catalysis, Schiff base formation Example: chymotrypsin
  • 18. 4) Catalysis by bond strain Example: lysozyme Substrate, bound substrate, and transition state conformations of lysozyme . The substrate, on binding, is distorted from the typical 'chair' hexose ring into the 'sofa' conformation, which is similar in shape to the transition state.
  • 19. 5) Catalysis by proximity Similar reactions will occur far faster if the reaction is intramolecular.
  • 20. Traditionally, enzymes were simply assigned names by the investigator who discovered the enzyme. As knowledge expanded, systems of enzyme classification became more comprehensive and complex. Currently enzymes are grouped into six functional classes by the International Union of Biochemists (I.U.B.). Number Classification Biochemical Properties 1 Oxidoreductases Act on many chemical groupings to add or remove hydrogen atoms. 2 Transferases Transfer functional groups such as methyl or glycosyl between donor and acceptor molecules. Kinases are specialized transferases that regulate metabolism by transferring phosphate from ATP to other molecules. 3 Hydrolases Add water across a bond such as C-C, C-O, C-N, P-O, hydrolyzing it. 4 Lyases Add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bonds. 5 Isomerases Carry out many kinds of isomerization: L to D isomerizations, mutase reactions (shifts of chemical groups) and others. 6 Ligases Catalyze reactions in which two chemical groups are joined (or ligated) with the use of energy from ATP.
  • 21.
    • ENZYME CLASSES Enzymes are also classified on the basis of their composition. There are two types :
    • Simple enzymes - composed wholly of protein
    • Complex enzymes - composed of protein plus a relatively small organic molecule.
    • Complex enzymes are also known as holoenzymes . In this terminology the protein component is known as the apoenzyme , while the non-protein component is known as the coenzyme or prosthetic group where prosthetic group describes a complex in which the small organic molecule is bound to the apoenzyme by covalent bonds; when the binding between the apoenzyme and non-protein components is non-covalent, the small organic molecule is called a coenzyme . Many prosthetic groups and coenzymes are water-soluble derivatives of vitamins . It should be noted that the main clinical symptoms of dietary vitamin insufficiency generally arise from the malfunction of enzymes, which lack sufficient cofactors derived from vitamins to maintain homeostasis.
  • 22.  
  • 23.  
  • 24. Simmons C R et al. J. Biol. Chem. 2006;281:18723-18733 The cytosolic enzyme cysteine dioxygenase (CDO) (EC 1.13.11.20) catalyzes the irreversible oxidation of cysteine to cysteine sulfinate
  • 25. Overall structure of Cysteine dioxygenase. Simmons C R et al. J. Biol. Chem. 2006;281:18723-18733
  • 26. Electron density evidence for key features of the CDO active site. Simmons C R et al. J. Biol. Chem. 2006;281:18723-18733
  • 27. The non-protein component of an enzyme may be as simple as a metal ion or as complex as a small non-protein organic molecule. Enzymes that require a metal in their composition are known as metalloenzymes if they bind and retain their metal atom(s) under all conditions with very high affinity. Those which have a lower affinity for metal ion, but still require the metal ion for activity, are known as metal-activated enzymes. Metals Zn, Fe, Ni, Co are common.
  • 28. Simmons C R et al. J. Biol. Chem. 2006;281:18723-18733 Metal coordination comparison between the R. norvegicus (A) and M. musculus (B) CDO models. A, tetrahedrally coordinated iron center in native R. norvegicus CDO. The CDO iron (orange sphere) is coordinated by the Nϵ2 atoms of three histidine ligands (His86, His88, and His140) and water molecule Wat4 (red sphere) bound to the catalytic iron. There are 3 water molecules Wat 160, Wat 161 and Wat 162 coordinated with hexacoordinated Ni center (green sphere) in M. musculus . iron nickel
  • 29. Enzymes have optimum temperature and optimum pH
  • 30. pH optimum of some important enzymes
  • 31. Enzymes in diagnosis of pathology
  • 32. LDH isozyme pattern in mammalian (mouse) tissues by polyacrylamide gel electrophoresis The measurement of LDH is especially diagnostic for myocardial infarction because this enzyme exist in 5 closely related, but slightly different forms (isozymes).
  • 33.
    • Time following a myocardial infarct
    • within 24-48 h - serum LDH levels rise
    • between 2-3 d - peak in serum LDH
    • within 5-10 d - return to normal or
    • pre-injury level
    • provided no further
    • injury occurs
    • Especially diagnostic is a comparison of the LDH-1/LDH-2 ratio. Normally, this ratio is less than 1. A reversal of this ratio is referred to as a " flipped LDH ". Following an acute myocardial infarct the flipped LDH ratio will appear in 12–24 hours and is definitely present by 48 hours in over 80% of patients. Also important is the fact that persons suffering chest pain due to angina only will not likely have altered LDH levels.
  • 34. Isozymes of some important enzymes used in diagnosis
  • 35.
    • This test is done if a CPK test reveals that your total CPK level is elevated. CPK isoenzyme testing can help pinpoint the exact soure of the damaged tissue.
    • CPK is made of three slightly different substances:
    • CPK-1 (also called CPK-BB) is found mostly in the brain and lungs
    • higher than normal result in brain cancer, brain injury
    • CPK-2 (also called CPK-MB) is found mostly in the heart
    • Higher than normal result in heart attack
    • CPK-3 (also called CPK-MM) is found mostly in skeletal muscle
    • Higher than normal result in crush injury in muscle and
    • muscular dystrophy
    •          
    Creatine phophokinase - CPK
  • 36. The typical enzymes measured in the serum are 1) ALT (alanine aminotransferase or serum glutamate pyruvate transaminase, SGPT). 2) AST (aspartate aminotransferase or serum glutamate oxaloacetate transaminase, SGOT) ALT is found predominantly in hepatocytes. It is diagnostic of liver disease or viral hepatitis . When assaying for both ALT and AST the ratio of the level of these two enzymes can also be diagnostic. Normally in liver disease or damage that is not of viral origin the ratio of ALT/AST is less than 1. However, with viral hepatitis the ALT/AST ratio will be greater than 1. Measurement of AST is useful not only for liver involvement but also for heart disease or myocardial infarction. The level of AST elevation in the serum is directly proportional to the number of cells involved as well as on the time following injury that the AST assay was performed. Serum aminotransferases
  • 37.
    • Time following injury
    • within 8 h - AST levels rise
    • 24-36 h - peak in AST level
    • within 3-7 d - AST levels return to normal or pre-injury level provided no further injury occurs
    • Although measurement of AST is not, in and of itself, diagnostic for myocardial infarction, taken together with LDH and CK measurements, the level of AST is useful for timing of the infarct.
  • 38. THANK YOU! Living to 100 : Major-gene mutants to long life telomerase story

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