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Enzyme Dialogue

Enzyme Dialogue . How to decipher the meaning of enzyme terms. Enzyme Activity. Definition, Meaning and Measurement of . What is an Enzyme?. An enzyme is a protein that behaves as a catalyst. How do we know how much enzyme is present in a cell, a tissue, an extract, etc.?.

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Enzyme Dialogue

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  1. Enzyme Dialogue How to decipher the meaning of enzyme terms

  2. Enzyme Activity Definition, Meaning and Measurement of ...

  3. What is an Enzyme? An enzyme is a protein that behaves as a catalyst How do we know how much enzyme is present in a cell, a tissue, an extract, etc.? We measure the amount of enzyme by measuring its activity. Come Again! An enzyme is measured by what it does not by what it is. Why is it done that way? Because most of the time we never have a pure enzyme. Therefore, we measure its amount indirectly by its activity

  4. What do you mean by activity? Activity is a velocity. We measure the rate of change of of a specific amount of substrate per unit time. What are the conditions? We must assume the enzyme is limiting. Therefore, the substrate must be in abundant supply. How do you turn activity into enzyme? We do this by assigning a unit of activity and relate that to the enzyme. What is a Unit? A unit is the amount of enzyme that catalyzes the change of one micromole of substrate in one minute.

  5. How does that tell me how much enzyme is present? If we relate enzyme units to micrograms of enzyme, we can immediately determine the weight of enzyme by knowing the activity. What if the enzyme is not pure? We then relate activity to mg of protein present. That measurement gives us the specific activity. What if the enzyme is pure? Then we can express activity as a turnover number. What’s that? Turnover number is a time-dependent change expressed as moles of substrate changed per mole of enzyme present.

  6. Unit of Enzyme Activity Amount of enzyme sufficient to convert one micromole of substrate to product in one minute Example An extract catalyzed the change of 20 micromoles of substrate to product in 5 minutes. How many units of enzyme was present in the extract? ANSWER 4 UNITS OF ENZYME

  7. Specific Activity Units per mg of protein Example 5 mg of protein were in an extract that catalyzed the change of 100 micromoles of substrate to product in 10 minutes. What is the specific activity of the enzyme? Answer: 2 units per mg of protein

  8. E E 6 4 = = 10 10 E + P E + P Practical Meaning of Specific Activity Relates enzyme activity to protein. Gauges purity of an enzyme E P E P E E P E P E P P E P E E P P P E

  9. Specific Activity and Protein Purification

  10. Turnover Number Relates moles of substrate changes to moles of enzyme Present 1. Must have pure enzyme 2. Must know Molecular weight of enzyme T.O. is synonymous with Kcat, the catalytic constant.

  11. Enzyme Specificity

  12. 0 1 2 3 4 5 6 7 8 9 Factors that Effect Enzyme Activity Trypsin Sharp drop Pepsin Heat denaturation Activity 0 10 20 30 40 50 60 oC pH

  13. COFACTORS AND COENZYMES Organic

  14. Inorganic Cofactors

  15. O NH2 C NH2 N N O O O -P- H O O N N + CH2O-P-O-P-OCH2 N O O O O O Niacinamide (niacin) OH HO OH HO REDOX COFACTORS- NAD+, NADP Nicotine Adenine Dinucleotide (NAD+) Nicotine Adenine Dinucleotide phosphate (NADP+)

  16. Active site H H + H .. H + Reduced Coenzyme NADH + H+

  17. Pseudo glycosidic bond REDOX COFACTORS FAD H Ring holds 2 electrons and 2 protons .. H Flavine adenine dinucleotide FADH2 FAD

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