Department of Biochemistry and Molecular Biology, New Jersey Medical School

1
ENZYMES-Activity Measurements

• Enzyme and substrate form a complex
• k1 k3
• E S ES E
  P
• k2

2
VELOCITY vs. S CONCENTRATION

• Initial Velocity increases with increase in S
  and then becomes independent of S

3
MICHAELIS-MENTEN EQUATION

• Based upon the formation of ES complex, following
  equation is derived
• Vmax S
• v Km S
• Vmax is maximal velocity when enzyme is saturated
  with the substrate.
• Km is Michaelis constant and in many cases is
  equal to the dissociation constant of ES complex

4
Km and Vmax

• Km
• Is a ratio of rate constants k2 k3/k1
• Is equal to S when initial rate(v) is equal to
  ½ Vmax
• Is a property of ES complex does not depend on
  the concentration of E or S
• Vmax
• Maximum velocity at a fixed E concentration
• Directly proportional to the E

5
DETERMINATION OF Km AND Vmax

• MM Equation can be rearranged to
• 1 Km x 1 1
• V Vmax S Vmax

6
MEASUREMENT OF ACTIVITY IN CLINICAL SAMPLES AND
FLUIDS

• Vmax directly proportional to E so make
  activity measurements at saturating S. To do so
  have S 10Km
• And then v 10/11 Vmax

Vmax
E
7
PHYSIOLOGICAL IMPORTANCE OF Km IN GLUCOSE
HOMEOSTASIS

• Plasma Glucose 100 mg 5 mM
• Brain Glucose 2 mM
• Km for glucose in liver for glucokinase 15 mM
• Km for glucose in brain for hexokinase 0.01 mM
• Km for ATP for both liver and brain enzymes 0.1
  mM
• At 1.0 mM ATP in cells, glucose will be oxidized
  in liver at 25 of Vmax
• In brain at 100 of Vmax
• Please Calculate?

8
Treatment of Leukemia

• PML patients have high plasma Asparagine(Asn)
• Concentration (50 uM). The objective is to lower
  this
• concentration to 5 uM.
• Asn H2O -------------gt Asp NH3
• asparaginase
• Source Km (uM) Vmax Vmax/Km
• Bovine 200 50 0.25
• Rat 40 30 0.75
• Bacteria 1 30 30
• The ratio Vmax/Km is referred to as efficiency of
  catalysis.

9
ENZYME-Kinetics

• Learning Objectives
• How do enzyme catalyzed reactions differ from
  other catalyzed reaction?
• What are the assumptions in the derivation and
  what is the significance of MM equation?
• How can one determine Km and Vmax?
• What is the significance of Km and Vmax?
• What conditions are used in clinical analyses?
• How can the knowledge of Km values be used to
  explain physiological function?
• Is the knowledge of Km values useful in designing
  drugs?