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Lecture 14

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Department
Biology
Course
BIOL 425
Professor
Mark Shrimpton
Semester
Fall

Description
Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity, in enzyme units.Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. Enzyme activity as given in katal generally refers to that of the assumed natural target substrate of the enzyme. Enzyme activity can also be given as that of certain standardized substrates, such as gelatin, then measured in gelatin digesting units (GDU), or milk proteins, then measured inmilk clotting units (MCU). The units GDU and MCU are based on how fast one gram of the enzyme will digest gelatin or milk proteins, respectively. 1 GDU equals approximately 1.5 MCU. The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min mg ). Specific activity gives a measurement of the activity of the enzyme. It is the amount of product formed by an enzyme in a given amount of time under given conditions per milligram of total protein. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of total protein. The SI unit is katal kg , but a more practical unit is μmol mg min . Specific activity is a measure of enzyme processivity, at a specific (usually saturating) substrate concentration, and is usually constant for a pure enzyme. For elimination of errors arising from differences in cultivation batches and/or misfolded enzyme etc. an active site titration needs to be done. This is a measure of the amount of active enzyme, calculated by e.g. titrating the amount of active sites present by employing an irreversible inhibitor. The specific activity should then be expressed as μmol min mg active enzyme. If the molecular weight of the enzyme is known, the turnover number, or μmol product sec μmol of active enzyme, can be calculated from the specific activity. The turnover number can be visualized as the number of times each enzyme molecule carries out its catalytic cycle per second. The rate of a reaction is the concentration of substrate disappearing (or product produced) per unit time The % purity is 100% × (specific activity of enzyme sample / specific activity of pure enzyme). The impure sample has lower specific activity because some of the mass is not actually enzyme. If the specific activity of 100% pure enzyme is known, then an impure sample will have a lower specific activity, allowing purity to be calculated. All enzyme assays measure either the consumption of substrate or production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains appr
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