Enzymes Kinetics

1. Properties
   1. Fundamental propterties of proteins
      1. 1¡, 2¡, 3¡, maybe 4¡ level of structure
      2. Heat labile
      3. Vesicular transport across membranes=huge
   2. Accelerate reactions by lowering activation energy
      (NOT by altering amount of product made!)
   3. Forms activated complex: E + S <-> ES <-> E + P
   4. Not used up ^ ^
   5. Effective at low concentration
   6. Specific to substrate used, reaction catalyzed, product made
2. Enzyme Kinetics: Michaelis-Menten plot:

   

   

   For this plot Km is the Substrate Concentration at 1/2 maximum rate.

   The value of is the "affinity" of the enzyme for the substrate

   How do we get Vmax and Km?

   • Inspection:

     The eye-brain combination is a good integrator!

     No statistical measures possible!

   • Graphic method:

     Do some algebra on Michaelis-Menten equation:

     Now if we let: and then similar to y = mx + b

     Which plots to a straight line with: and

     Lineweaver-Burke Plot:

     

     

     This plot lets us determine Vmax and Km with much less "subjectivity."

     Vmax^-1 = y intercept
     -Km^-1 = x intercept

     Statistical Method: linear regression of transformed data

   • Statistical Method: nonlinear regression

     This is the BEST way to do it as it uses the Michaelis Menten function directly to fit the curve to the data.

Competitive Inhibitors decrease substrate affinity (therefore increase Km) but do not alter Vmax so the Lineweaver Burke plots in presence and absence have altered slope but have same y-intercept.

Noncompetitive inhibitors will alter the Vmax and so the y-intercept will be different.

In lab tomorrow we will try to test out some inhibitors on the function of polyphenol oxidase:

Here are some possible competitive inhibitors.

For non-competitive inhibitors we could try:

KAs-binds to SH groups of amino acids

KCN-chelates iron and copper

PTU-chelates copper

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