lecture 3: enzyme kinetics

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  • 1. Enzyme Kinetics Lab C1 Two periods Pages 65-91

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
    • Measurement accuracy
    • Spectrophotometry
      • Relationship between concentration and absorbance
  • 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 chemical reactions which are thermodynamicallyveryunfavorable
    • Break and form covalent bonds
    • Move large structures
    • Effect three dimensional structure
    • Regulate gene expression
  • Do so through catalysis

4. Effect of enzymes

  • A common biological reaction can take place without enzyme catalysis
    • Will take 750,000,000 years
  • With enzyme 22 milliseconds
  • Even improvement of a factor of 1,000 would be good
    • Only 750,000 years
    • Living systems would be impossible

5. Sucrose

  • A bag of sugar can be stored for years with very little conversion to CO 2and H 2 O
    • This conversion is basic to life
    • When consumed is converted to chemical energy very fast

6. Catalysis

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

7. Enzymes

  • Have immense importance in a number of fields.
    • Genetic diseases arefrequently 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

8. Enzymes

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

9. 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 choosea career in biochemistry, molecular biology, biophysics, biochemical genetics, pharmacology, cell biology, etc

10. Kinetics

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

11. StickaseFrom Lehninger; third edition 12. 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

13. Rate Constant: k

  • AB
  • Velocity of Rx
    • V=d[B]/dt
      • d=change
    • V=-d[A]/dt
  • V=d[B]/dt = -d[A]/dt = k[A]
  • Large k rapid Rx
  • Small k slow Rx

14. Catalysis

  • Simple reactionA[s] B[P]
  • E + SESE + P
  • At steady state
  • [ES] = (k 1 /k -1+ k 2 ) [E] [S]
  • K 2also known as k cat

k -1 k 2 k 1 15. k m : A ratio of Rate constants page 71-74

  • [ES] = (k 1 /k -1+ k 2 ) [E] [S]
  • k m = k -1+ k 2 / k 1
    • K m=Michaelis constant

16. Initial velocityV o

  • 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 rate of reaction, V oas [S] increases
    • Until ratebecomes constant: approaches V max

17. Effect of [Substrate] 18. Effect of [substrate] on R XVelocity V max= k 2[E] t [E] t=total enzyme =[E] +[ES] 19. Michaelis-Menten Equation V 0=V max[S] K m+[S] 20. Lineweaver-Burk Plot

  • K m1
  • V 0 V max[S]V max

21. Can calculate K m

  • One of the most important descriptive terms in all of biology

22. Lineweaver-Burk Plot

  • K m1
  • V 0 V max[S]V max

23. Alcohol Dehydrogenase: ADH

  • CH 3 CH 2 OH + NAD +CH 3 CH 2 O+H +
  • +NADH
  • Catalyses conversion of ethanol to aldehyde using co-enzyme NAD+
    • MH2 + NAD+ -> NADH + H+ + M: + energy, where M is a metabolite.
  • NAD+ oxidized to NADH reduced

24. NAD+ to NADH 25. NAD+ 26. NADH 27. Reaction is complex

  • ADH +ALCADH-ALC
  • ADH + NADADH-NAD
  • ADH-NAD +ALCADH-NAD-ALC
  • We are not looking at this

28. Alcohol Dehydrogenase CH 3 CH 2 OH + NAD +CH 3 CH 2 O+H + + NADH We will measure the forward Rx (k2 )as increased absorbanceat 340. Only NADH absorbs at this wave length (page 70) Will find the assay conditions which produce max activity K m 29. 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

30. Measure V owith increasing [S] Re-plot these data in thedouble-reciprocalLineweaver-Burk plot 31. This Lab and Next Lab

  • Part one Kinetic Curve, V 0Lineweaver-Burk page 76-78
    • Determine basic properties of enzyme K M
  • Part two Page 78-80
    • Effects of concentration, temperature and pH
  • Lab requirements: Page 90-91.

32. Experiment 1: Page 73&76 Kinetic curve.Add enzyme 33. Data table Page 77 Table C.1-1.____ Assay # Water (ml) Buffer Ethanol [S] NAD + ADH V 1/V 1/[S] 1 0.000 0.700 2.100 0.100 0.100 2 0.600 0.700 1.500 0.100 0.100 3 1.100 0.700 1.000 0.100 0.100 4 1.600 0.700 0.500 0.100 0.100 5 1.900 0.700 0.200 0.100 0.100 6 2.000 0.700 0.100 0.100 0.100 7 2.050 0.700 0.050 0.100 0.100 8 2.080 0.700 0.020 0.100 0.100 9 2.090 0.700 0.010 0.100 0.100 10 2.095 0.700 0.005 0.100 0.100 34. Be careful

  • 15 sec and 45 sec
    • Read same and low =
      • too little substrate
      • Didnt 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

35. Initial Velocity (page 75) 36. Sample data

  • Kinetic curveFigure C.1-5
  • Lineweaver-Burke Plot Figure C.1.6

37. This Lab

  • 2 Lab periods
  • Pre Labs 6 points
  • Lab Report 20 points

38. Clean up and Check out Page 89

  • 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

39. Kinetics Write Up

  • See report outline Page 90
  • Remember describe what happened in your experiment

40. Next time Examine the effects of: Temperature pH Substrate concentration 41. Next Exercise

  • Effects of Temp, pH and Enzyme concentration. Page