BC basic - from big bang

1
Enzyme Kinetics
Student A
Enzyme activity
Score
Student B
Student C
0 1 2 3 4
0 1 2 3 4
Substrate concentration
Exam Chapters
Increase the substrate concentration, observe the
change of enzyme activity
Juang RH (2004) BCbasics
2
Invertase (IT)
Reducing sugars
Non-reducing sugar
Reducing Power
IT
Sucrose
Fructose
Glucose

b
b
1 2 3 4 5 6
H2O
Juang RH (2004) BCbasics
3
Increase Substrate Concentration
S E ? P
(in a fixed period of time)
Juang RH (2004) BCbasics
4
Essential of Enzyme Kinetics
Steady State Theory
E
E


P
S
In steady state, the production and consumption
of the transition state proceed at the same rate.
So the concentration of transition state keeps a
constant.
Juang RH (2004) BCbasics
5
Constant ES Concentration at Steady State
S
P
ES
E
Juang RH (2004) BCbasics
6
An Example for Enzyme Kinetics (Invertase)
1) Use predefined amount of Enzyme ? E
2) Add substrate in various concentrations ? S
(x ?)
3) Measure Product in fixed Time (P/t) ? vo (y ?)
4) (x, y) plot get hyperbolic curve, estimate ?
Vmax
5) When y 1/2 Vmax calculate x (S) ? Km
Vmax
1/2
Juang RH (2004) BCbasics
Km
Double reciprocal
Direct plot
7
A Real Example for Enzyme Kinetics
Data
v (mmole/min)
S
Absorbance
1/S
1/v
no
0.25 0.50 1.0 2.0
0.42 0.72 0.80 0.92
0.21 0.36 0.40 0.46
4 2 1 0.5
2.08 1.56 1.35 1.16
1 2 3 4
? ? ? ?
(1) The product was measured by spectroscopy at
600 nm for 0.05 per mmole (2) Reaction time was
10 min
Direct plot
Double reciprocal
Juang RH (2004) BCbasics