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Enzymes

Enzymes. GENERAL FEATURES Enzymes are biological catalysts that speed up biochemical reactions. GENERAL FEATURES Enzymes are biological catalysts that speed up biochemical reactions. They: a) do not alter the ΔG of the reaction they catalyse but.....

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Enzymes

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  1. Enzymes

  2. GENERAL FEATURES • Enzymes are biological catalysts that speed up biochemical reactions.

  3. GENERAL FEATURES • Enzymes are biological catalysts that speed up biochemical reactions. • They: a) do not alter the ΔG of the reaction they catalyse but..... b) ..... they lower its activation energy and c) .... enhance the rate of the reaction.

  4. Nearly all enzymes are proteins although some RNA molecules have been shown to have catalytic activity. • Enzymes vary widely in size and structure. • They are (usually!) highly specific for the reaction that they catalyse. • The rates at which someenzymes operate are subject to regulation and control.

  5. CATALYTIC POWER • This is the degree to which an enzyme speeds up a reaction. • The enzyme reaction rate can increase many times compared to the uncatalysed rate ( in the range 103-1017 times)

  6. An example: CO2 + H2O H+ + HCO3- carbon dioxideCAbicarbonate ions CA - carbonic anhydrase (present in red blood cells) - increase reaction rate ~107 times • Without this catalytic power tissues would not be able to get rid of toxic CO2 rapidly enough.

  7. SPECIFICITY • Each type of enzyme will only catalyse one particular chemical reaction (or set of closely related reactions) • Enzymes are specific with regard to substrate (reactants). • Note: this is generally true but some enzymes are able to accept a wide variety of substrates egcytochrome P450 • This substrate is catalysed at the active siteof the enzyme.

  8. The Koshland induced fit model of enzyme catalysis

  9. An example of specificity:

  10. Product specificity: • With the same substrate, different enzymes catalyse the formation of different product. • Therefore different enzymes can effectively direct (or ‘funnel’) the same substance into different metabolic pathways.

  11. NOMENCLATURE (or NAMING) OF ENZYMES • Enzymes are generally named after the specific reactions they catalyse. • Generally all should end in “-ase”, but (for historical reasons) there are some exceptions egtrypsin, papain, bromelain. • Enzymes can be defined unambiguously by their EC number (EC = Enzyme Commission). ~2000 have been classified in this way. • Many enzymes have common names as well as systematic names.

  12. eg carbonic anhydrase (this is the common name) = carbonate hydro-lyase (this is the systematic name) = EC 4.2.1.1 (this is the Enzyme Commission number).

  13. ENZYME MEASUREMENT • How do we measure enzymes? In fact we measure their activitynot their mass or concentration. • By carrying out an enzyme assay (assay means measurement) that measures the rate of the reaction catalysed. To assay an enzyme: • take the enzyme (either pure or not), • add components needed for chemical reaction (substrates), • use appropriate conditions (pH, temperature, cofactors) • follow reaction over time (rate of reaction).

  14. To calculate the rate of reaction plot a graph of the extent of reaction ( either formation of products or disappearance of reactants) against time. This is called a Reaction-Time Plot or Progress Curve.

  15. mmol minutes

  16. POINTS TO CONSIDER • How can you be sure that the reaction observed is enzyme catalysed ( and not just occurring at that rate anyway)?

  17. POINTS TO CONSIDER • How can you be sure that the reaction observed is enzyme catalysed ( and not just occurring at that rate anyway)? • Ans. By including a control experiment - the enzymeblank. This contains everything except enzyme. Usually these blanks show little or no measurable conversion of substrate.

  18. Why does the enzyme-catalysed reaction ‘tail- off’ with time?

  19. Why does the enzyme-catalysed reaction ‘tail- off’ with time? • Ans. For several possible reasons: • Enzyme becomes thermally inactivated with time • Product inhibition. • Substrate depletion. • Product build-up  reverse reaction.

  20. Because of this ‘tailing-off’, the reaction rate varies. The rate is actually measured in the early stages of the progress curve where the above factors have less influence. This rate is called the initial rate or initial velocity (Vo) and is expressed as μmol substrate converted to product(s) per minute.

  21. It is more useful to express enzyme rate not as initial rate but as specific activity. Specific activity = μmol substrate converted per minute per mg protein. = μmol min-1mg-1 Why? Ans. Because it gives information on purity of enzyme. The higher the specific activity the greater the purity.

  22. FACTORS AFFECTING ENZYME ACTIVITY TEMPERATURE • For many reactions Q10 = 2, which means that for a 10 C rise in temperature the rate doubles. • This is true for enzyme reactions, except that if the temperature is held above the optimum temperature for too long a period then the enzyme inactivates. • NOTE: the optimum temperature varies according to species and time of exposure!

  23. Enzymes become inactivated at higher temperatures because they are proteins • An enzyme may return to normal activity on cooling but only if it is able to renature.

  24. pH All enzymes are sensitive to pH, each enzyme having a particular optimum pH at which catalytic activity is maximum. Enzymes are usually assayed (measured) at optimum pH. The optimum is maintained by using a buffered solution .

  25. Many mammalian enzymes have an optimum pH = 7.4 but pepsin has an optimum of pH=2. Why?

  26. Many mammalian enzymes have an optimum pH = 7.4 but pepsin has an optimum of pH=2. Why? Ans. Because pepsin is a protease enzyme which operates in the stomach where the pH is low.

  27. Proteins contain a large number of ionisable groups (due to side chains which carry a charge). The ionisation depends on the pKa of the ionisable group and the pH. • Altering the pH alters the degree of ionisation. • If one or more groups at the active site must be ionised to permit catalysis then altering the pH will affect the activity of the enzyme.

  28. COFACTORS • Some enzymes contain one or more parts which are vital to enzyme activity. These are coenzymes, cofactors or prosthetic groups. • Their role is to aid catalysis. They often react with the substrates and they are located at the active sites of enzymes.

  29. CONCENTRATION - RATE CURVES If the initial rate is measured for progress curves at different [substrate] then a plot of concentration against rate can be obtained. The Relationship between Rate of Reaction (Vo) and the Substrate Concentration [S] for an Enzyme-Catalysed Reaction

  30. The curve never reaches Vmax ( this is because a rectangular hyperbola is asymptotic!) • The concentration-rate curve is described by the following equation

  31. Km is the Michaelis constant . = a measure of affinity of that substrate for the enzyme - the lower the Km the greater the affinity • Because Vmax is never reached it can only be estimated from the concentration-rate curve • Vmax and Km are key paramemeters in defining how an enzyme functions. • How do we determine them?

  32. Vo = Vmax[S] can be transformed by Km + [S] taking reciprocals. This allows us to plot the curve as a straight line in the form y = mx + c

  33. Vo = Vmax[S] Km + [S] 1 = Km + [S] Vo Vmax[S]

  34. Vo = Vmax[S] Km + [S] 1 = Km + [S] Vo Vmax[S] 1 = Km + [S] Vo Vmax[S] Vmax[S]

  35. Vo = Vmax[S] Km + [S] 1 = Km + [S] Vo Vmax[S] 1 = Km + [S] Vo Vmax[S] Vmax[S] Plotting 1 against 1 gives a straight line.Vo [S]

  36. A Lineweaver Burk plot

  37. Note that it is not the only transformation possible. For example, the Hofstee-Eadie plot which arises from plotting Vo against Vo [S] • Using these transformations , Km and Vmax can be determined easily and accurately.

  38. INHIBITION • Two broad classes: irreversible and reversible. • Irreversible inhibition cannot be relieved by removing inhibitor - the enzyme is ‘poisoned’. • Reversible inhibition can be relieved by removal of the inhibitor from solution.

  39. Reversible inhibitors can be subdivided a) competitive b) non-competitive c) uncompetitive

  40. COMPETITIVE INHIBITION (CI) • Consider that there exists a molecule which resembles the substrate for an enzyme and the enzyme accepts it at the active site. • There are 2 possibilities: i) the molecule is converted to a product - then it is a competing alternative substrate . ii) the molecule does not undergo catalysis - then it is a competitive inhibitor.

  41. Substrate Competitive inhibitor

  42. In other words a competitive inhibitor competes with the substrate for the active site.

  43. In other words a competitive inhibitor competes with the substrate for the active site. +CI Vo [S]

  44. Note that 1 unchanged  Vmax is unchanged Vmax But: -1 changed -1  Km changed Km Kmapp

  45. Km increases with increasing [Inhibitor] since more inhibitor occupies the active site effectively changing the affinity of the active site for the substrate. • As [S] increases Vo  Vmax since if [S] very large compared with [In], then S outcompetes In.

  46. NON-COMPETITIVE INHIBITION (NCI) Active site

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