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Biochemistry Lab

Biochemistry Lab. Enzyme Kinetics. Steady State Approximation. Steady State Approximation. Steady State Approximation. Assumption 1: k -1 >> k 2 Rational: An equilibrium between Enzyme and Substrate is achieved prior to Product formation. Assumption 2: Rational:

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Biochemistry Lab

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  1. Biochemistry Lab Enzyme Kinetics

  2. Steady State Approximation

  3. Steady State Approximation

  4. Steady State Approximation Assumption 1: k-1 >> k2 Rational: An equilibrium between Enzyme and Substrate is achieved prior to Product formation Assumption 2: Rational: Except the initial and final stages of reaction, the concentration of ES does not significantly change.

  5. Steady State Approximation

  6. Steady State Approximation Assumption 1: k-1 >> k2 Rational: An equilibrium between Enzyme and Substrate is achieved prior to Product formation Assumption 2: Rational: Except the initial and final stages of reaction, the concentration of ES does not significantly change.

  7. Steady State Approximation distribute Rearrange Rearrange

  8. Steady State Approximation Assumption 1: k-1 >> k2

  9. Steady State Approximation

  10. Steady State Approximation Applications of M-M Kinetics Turnover Number (kcat)  reports on number of processes per enzyme Catalytic Efficiency ( )  Apparent 2nd order rate constant

  11. Determining KM and Vmax Lineweaver-Burk Relationship

  12. Hydrolysis of Polygalacturonic Acid How can we monitor this reaction? PectateLyase

  13. Experimental Details Measure the initial velocity at 6 different substrate concentrations Experimental Buffer: 50 mMTris-HCl, 0.1 mM CaCl2, pH 8.5 Why is the Calcium present? PectateLyase 50-100x dilution in experimental buffer What happens if the enzyme is too concentrated? Substrate  Prepare 6 PGA sample concentrations from 5% PGA stock Make extra! What should you use as an experimental blank?

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