Basic enzymology modified


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Basic enzymology modified

  1. 1. Enzymes are protein catalysts responsible for most of the chemical reactions of the bodyThey are found in: cells and all tissues; serum to which gain access from injured cells, cells undergone stress
  2. 2. in disease states, caused in increased membrane permeabilitycaused by increase rates of intracellular synthesissubsequent diffusion of enzymes Enzymes found in serum will help physicians diagnose certain disease and aid in the monitoring of the disease condition
  3. 3. Increase serum levels increased release of enzyme from source o necrosis o increased membrane permeability increased size of tissue source of enzyme impaired excretion of enzyme increased enzyme synthesis
  4. 4. Decreased serum levels decreased formation - Genetic - Acquired - Enzyme inhibition poisoning lack of cofactors
  5. 5. Alkaline phosphatase – diagnose osseous and hepatobiliary diseasesAcid phosphatase – in the diagnosis of prostate cancersAmylase and lipase – diagnosis of pancreatic diseaseSGPT – liver diseases (hepatitis)SGOT – cardiac and liver diseases
  6. 6. Catalyst A substance that enhances the rate of a chemical reaction but is not permanently altered by the reaction Decreases the activation of energy required for a chemical reaction and provides an alternative reaction pathway that requires less energy
  7. 7. Enzymes are neither produced or consumed in the reactionEnzymes do not cause the reaction but enhances the reaction to occurEnzymes are long sequences of amino acid
  8. 8. Enzymes are highly specific and produce only the expected product from given reactant or substrateEnzymes acts on moderate pH and temperature
  9. 9. Enzyme possess the following: Active site – where the substrate fits before converted to corresponding product Allosteric site – which binds effector molecules or modifiers which regulates enzyme activity
  10. 10. Apoenzyme – are protein portion of the enzymeCoenzyme – non-protein component; main participant during reaction with a substrate
  11. 11. help in enzymatic activity by orienting properly the substrate with the active site.Coenzyme – organic cofactor: NADH, NADPH, FAD and FMNActivator - usually metallic ions tightly bound to enzymes: Mg, Zn++, Cu++
  12. 12. Other enzymes are pro-enzymes or zymogens that are inactive but once released to their target sites they become activated.Isoenzymes – enzymes with similar enzymatic activities but differ in their chemical structure and origins
  13. 13. They are unchanged during the course and termination of chemical reaction.They demonstrate great specificity: Absolute – pyruvate kinase Group - phosphatases Bond - hydrolases Stereospecificity - transaminases
  14. 14. Enzyme binds to a single type of substrate because of complementary structures of the active site and substrateSubstrate’s overall shape and charge allows to interact with enzymes active site
  15. 15. Flexibility structure of protein is taken into accountSubstrate does not precisely fit into the rigid active site instead a non-covalent interaction of enzyme-substrate change it thus conforming to the shape of active site to the shape of the substrate
  16. 16. Substrate ConcentrationMichaelis-Menten hypothesis: the rate of substrate conversion to product is determined by  substrate concentration and  rate of dissociation of enzyme substrate complex first-order kinetics  At constant enzyme concentration, the velocity or speed of the enzyme reaction initially increases as the substrate concentration increases. Reaction rate is dependent on the substrate concentration
  17. 17. Zero-order kinetics The point at which the further addition of substrate does not anymore change the velocity of enzyme reaction. The reaction is now dependent on the enzyme concentrationKm Represents the substrate concentration where the velocity is ½ the maximum Represents the substrate concentration at which the enzyme yields half the possible maximum velocity It is also the measure of affinity of the enzymes with its substrate
  18. 18. Enzyme concentration The higher the enzyme concentration the higher is the reaction rate. (true only in zero-order kinetics)pH optimal pH varies with each enzyme
  19. 19. Temperature most denatures at 60oC usually optimum at 37oC there is a characteristic increase in the reaction rate for every 10oC increase before denaturation
  20. 20. Cofactors metals – transition metals (Zn++. Cu++ and Fe++) – effective electophilesInhibitors Competitive (compete for active site) Non-competitive (binds with enzyme at place other than active site) Uncompetitive (binds with E-S complex)
  21. 21. Enzymes expressed in units that represent one of the following:Increased concentration of one of the products  substrate and coenzymeThe rate of change of any 3 unit is a measure of rate of reactionThe catalytic rate is proportional to its concentration at normal condition
  22. 22. MICHEALIS-MENTEN HYPOTHESIS k1 k3E + S  (ES)  E + P  k2where: k1 is rate constant for ES formation k2 is rate constant for ES dissociation k3 is rate constant for product formation and release from active site
  23. 23. International Union of Biochemistry (IUB)Classify and name enzymes according to the type of chemical reaction it catalyzesUsing the name of the substrate or group on which the enzymes acts and added by suffix “-ase”Examples:  Urease  hydrolyzes urea  Amylase  metabolizes starch/amylum  Phosphatase  acting on phosphate esters
  24. 24. Biochemical functions, indicating substrate, class of reaction catalyzed designated by individual identification numbersFor clarity, the raction is also identified (examples are carbonic anhydrase, D- amino acid oxidase and succinic dehydrogenase)
  25. 25. Systemic name – nature of the reaction catalyzed is associated with unique numercal code designationOf two parts: substrate(s) acted upon and a word ending with –ase indicating the reaction involvedExample: L-Lactate:NAD+ oxidoreductase Trivial or practical name – may be identical to the systemic name but is often a simplification of it – useful in everyday work
  26. 26. Example:EC  L-Lactate:NAD+ oxidoreductase  lactate dehydrogenaseEC denotes Enzyme CommisionFirst number defines the class to which the enzyme belongs
  27. 27. Assigned to 6 classes 1. oxidoreductases 2. transferases 3. hydrolaseses 4. lyases 5. isomerases 6. ligases
  28. 28. next two numbers indicate the subclass and sub- subclass to which the enzyme is assignedExample: may be differentiated from the amino transferring subclass of the phosphate – transferring category or the ethanol acceptor sub-class from that accepting acyl groupThe last number is the specific serial number given to the enzyme within its subclass
  29. 29. 1. oxidoreductases - catalyzed oxidation- reduction reactionsREDUCTION (addition of hydrogen to a double bond)OXIDATION (removal of a hydrogen from a molecule to leave a double bond)The hydrogen is transferred with the use of coenzymeL-lactate:NAD+ oxidoreductase catalyzed pyruvate + NADH + H+  lactate + NAD+ subclasses: dehydrogenases, oxidases, oxygenases, reductases, peroxidases, and hydroxylases
  30. 30. 2. transferases – catalyzed reactions that involve the transfer of groups from one molecule to another (amine or phosphate group)ATP:creatine N-phosphotransferase (creatine kinase) involves ATP + creatine  ADP + creatine phosphate trivial names include with prefix “trans”: transcarboxylases, transmethylases and transaminases
  31. 31. 3. hydrolases – catalyze reactions in which the cleavage of bonds is accomplished by adding waterAmylase (cleavage of –C-O-C- bonds in starch)Lipase (breaks down triglycerides to form glycerol and free fatty acids)Acid phosphatase and alkaline phosphatase (remove phosphate group from a variety of molecules) subclasses: esterases, phosphatases, peptidases
  32. 32. 4. lyases – (lysis means “splitting”)catalyze reactions in which groups are removed to form a double bond or are added to a double bond (C-C, C-S, and C-N bonds)Aldolase (EC, D-fructose-1,6-biphosphate-D- glyceraldehyde-3-phosphate lyase) which cleaves the 6- carbon molecule fructose-1,6-diphosphate to produce two 3-carbon compounds: glyceraldehyde-3-phosphate and dihydroxyacetone phosphate  subclasses: decarboxylases, hydratases, dehydratases, deaminases, synthases
  33. 33. 5. isomerases – (heterogenous group) catalyze several types of intramolecular rearrangementsWhere it involves in the conversion of one isomer to another with examples of transformation will include the change of cis to trans; L-form to D-form; aldehyde to ketonethe reactions are generally reversible
  34. 34. Triose phosphate isomerase (EC, D-glyceraldehyde-3-phosphate ketol- isomerase)In the glycolytic pathway, involves in the isomerization of glyceraldehyde-3- phosphate (aldehyde) to dihydroxyacetone phosphate (ketone) epimerases – catalyze the inversion of assymmetric carbon atoms mutases – catalyze the intramolecular transfer of functional group
  35. 35. 6. ligases – catalyze bond formation between two substrate molecules forming a larger moleculeImportant in the activation of amino acids – protein synthesis Energy is always supplied by ATP Aminoacyl-tRNA
  36. 36. 1. Substrate Measurement  This starts with a high substrate concentration1. Product Measurement  This starts with zero initial product level  This is more accurate
  37. 37. Endpoint analysis the reaction is initiated by addition of substrate and is allowed to proceed for a period of time measurement of substrate or product is done at the end of the reactionMultipoint assay this measures the change in the concentration of the indicator substance at several intervals during the course of the assay
  38. 38. Kinetic assay this involves measurement of change in concentration as a function of time closely monitored at short interval has advantage over the end point if the concentration of the substrate is sufficiently high in comparison to enzyme then rate of reaction will be proportional to the concentration of enzyme thus the amount of product formed in a given period of time would be proportional to the amount of active enzyme present, with all other factors remaining constant
  39. 39. Use of coupled reactions enzymatic activity is measured by coupling the activity with colorimetric reaction the colored product is measured spectrophotometrically
  40. 40. 1. HEMOLYSIS  May cause falsely elevated enzyme concentration1. ANTICOAGULANTS  Many anticoagulants cause adverse effects on enzyme inhibiton, therefore, serum is preferred over plasma1. LACTESCENT OR MILKY SERUM  May result in variable absorbance readings in spectrophotometry
  41. 41. Enzymes are stable at 6oC for at least 24 hours and at room temperature for lesser periodsFor prolonged storage, use -20oC or lowerCK must be kept at -70oCLD4 and LD5 – liver isoenzymes are inactivated at refrigerator temperature
  42. 42. That’s all folks… Have a great day ahead!