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Enzyme Kinetics

Enzyme Kinetics. Lab C1 Two periods Pages 73-104. Protein Chemistry. This begins a 6 day journey into the field of protein chemistry You will learn a set of basic tools and protocols which will be important in the successful outcome. You have practiced Measurement accuracy

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Enzyme Kinetics

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  1. Enzyme Kinetics Lab C1 Two periods Pages 73-104

  2. Protein Chemistry • This begins a 6 day journey into the field of protein chemistry • You will learn a set of basic tools and protocols which will be important in the successful outcome. You have practiced • Measurement accuracy • Spectrophotometry • Relationship between concentration and absorbance • Today we will do a basic experiment in enzymology which will prepare you for a protein purification.

  3. Enzymes • Living organisms must be able to carry out events which are thermodynamically very unfavorable • Break and form covalent bonds • Move large structures • Effect three dimensional structure • Regulate gene expression • Do so through use of Enzymes

  4. Effect of enzymes • A bag of sugar can be stored for years with very little conversion to CO2 and H2O • This conversion is basic to life • This common biological reaction can take place without enzyme catalysis • Will take 750,000,000 years • Even improvement of a factor of 1,000 would be good • Only 750,000 years • Living systems would be impossible • With enzyme 22 milliseconds

  5. Catalysis • Carried out by very highly specialized class of proteins: Enzymes • Specialized to perform specific chemical reactions • Specialized to work in specific environments

  6. Enzymes • Have immense importance in a number of fields. • Genetic diseases are frequently defects in enzymes or increased/decreased levels of enzymes • Important diagnostic tools • Drugs exert effects by interacting with enzymes • MAO inhibitors • Used in food processing and in chemical industry • Enzyme inhibitors are a foundation of biological weapons

  7. Enzymes • A major aspect of experimental biochemistry is the purification and characterization of proteins that are enzymes • Chemical characterization • Physical characterization

  8. In the next six laboratories • You will go through the basic protocols that are used to purify and characterize catalytic proteins • The basic procedures are ones which you will use the rest of your career if you choose a career in biochemistry, molecular biology, biophysics, biochemical genetics, pharmacology, cell biology, etc……………

  9. Kinetics • Is the science that describes the properties of a chemical reaction including those mediated by enzymes (catalysis) • Measures changes in the concentration of substrate and/or products of a reaction to determine the velocity of the reaction • Measures the effects of concentration, temperature, pH etc to characterize the properties of the enzyme catalyzing the reaction

  10. Stickase From Lehninger; third edition

  11. Enzyme Kinetics • An approach to understanding the mechanism of action of enzymes • An approach to understanding how mutations may effect function • An approach to understanding how changes in the physical and chemical environments change function

  12. Rate Constant: k • A B • Velocity of Rx • V=Δ[B]/Δt • V=-Δ[A]/Δt • V=Δ[B]/Δt = -Δ[A]/Δt = k[A] • Units are quantity/unit time • e.g. Moles/Second • Large k rapid Rx • Small k slow Rx

  13. Catalysis • Simple reaction A [s] B [P] • E + S ES E + P • K2 also known as kcat • At steady state • [ES] = (k1/k-1 + k2) [E] [S] k1 k2 k-1

  14. km:A ratio of Rate constantspage 80-81(Info Box 5) • [ES] = (k1/k-1 + k2) [E] [S] • km= k-1 + k2/ k1 • Km =Michaelis constant

  15. Initial velocity Vo • When enzyme is mixed with high concentration of substrate [S] reaction goes rapidly to steady state. • Does not allow characterization • Use low starting [S] and increase • Hold [enzyme] constant • Measure initial rate of reaction, Voas [S] increases • Until rate becomes constant: approaches Vmax

  16. Effect of [Substrate]

  17. Effect of [substrate] on RX Velocity

  18. Michaelis-Menten Equation Vmax [S] Km +[S] V0 =

  19. Lineweaver-Burk Plot Units of Km are concentration

  20. Can calculate Km • One of the most important descriptive terms in all of biology

  21. Alcohol Dehydrogenase: ADH CH3CH2OH + NAD+ CH3CH2O + H+ +NADH Catalyses conversion of ethanol to aldehyde using co-enzyme NAD+ NAD+ oxidized to NADH reduced

  22. NAD+

  23. NAD+ to NADH Absorbs at λ 340

  24. Reaction is complex • ADH +ALC ADH-ALC • ADH + NAD ADH-NAD • ADH-NAD +ALC ADH-NAD-ALC • We are not looking at this

  25. Alcohol Dehydrogenase CH3CH2OH + NAD+ CH3CH2O + H+ + NADH We will measure the forward Rx (k 2)as increased absorbance at 340. Only NADH absorbs at this wave length (page 70) Will find the assay conditions which produce max activity and calculate Km

  26. WHAT ARE WE MEASURING ? • Production of NADH • NAD+ NADH • Wavelength shift • Depends on participation of Alcohol and ADH • How can you do this • Ensure that NAD is not a rate limiting component. • [NAD] constant and high • [ADH] constant • [ETOH] low and increasing

  27. Measure Vo with increasing [S] Remember Vo= Δ NADH/Δ Time. Re-plot these data in the double-reciprocal Lineweaver-Burk plot

  28. This Lab and Next Lab • Part one Kinetic Curve (Figure C.1-5), V0 Lineweaver-Burk (Figure C.1-6) page 86-88 • Determine basic properties of enzyme KM • Part two Page 89-92 • Effects of concentration, temperature and pH • Report requirements: Page 102-104.

  29. Experiment 1: Page 86&76 Add enzyme Kinetic curve.

  30. Experiment 2 Page 87-88Determine Km and Vmax • Pipetting accuracy and timing is critical • Clean cuvette • Can check clean by adding all components except ADH and placing in spectrophotometer • Absorbance should not change with time

  31. Data table Page 87 Km

  32. Initial Velocity (Vo page 75)

  33. Be careful • 15 sec and 45 sec • Read same and low = • too little substrate • Didn’t add enzyme • Read same and high • Reaction is over • Contaminated one of your solutions with enzyme • Did not clean cuvette from previous assay • Remember you are determining the optimal conditions for this assay

  34. Sample data • Kinetic curve Figure C.1-5 • Lineweaver-Burke Plot Figure C.1.6

  35. This Lab • 2 Lab periods • Pre Labs 6 points • Lab Report 25 points

  36. Clean up and Check outPage 101-102 • Return pipetters to rack • Check that you have not left cuvette in spec • Clean any spill in spec • Clean & rinse the cuvette • Clean and rinse test tubes • Throw all waste in trash

  37. Kinetics Write Up • See report outline Page 102 • Remember describe what happened in your experiment

  38. Next time Examine the effects of: Temperature pH Substrate concentration

  39. Next Exercise • Effects of Temp, pH and Enzyme concentration. Page 89-92 • Read carefully “Factors that affect catalysis” (Page 93-101)prior to coming to lab. • Lab report on Enzyme Kinetics due at start of protein purification • Remember to find the Km of another enzyme and compare it to ADH

  40. Temperature Dependence page 94

  41. Effect of pH page 99

  42. Extra Credit for this Lab • At lower temperatures the kinetic rate change with temperature demonstrates Arrhenius behavior • Plot log Vo versus 1/T degrees Kelvin • Should result in a straight line • Slope = Ea (activation energy)/ R (Gas constant 1.9872041(18)×10−3 Kcal/mol

  43. Arrhenius plot Slope = _Ea/R R=1.9872041(18)×10−3 Kcal/mol

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