KINETIKA ENZIM

• Kinetika Enzim mempelajari kecepatan reaksi

enzimatis
• Studi kinetika berguna untuk :
- mengukur konsentrasi enzim dalam campuran
(dengan mengukur aktivitas katalitik)
- Mengukur katalitik effisiensi.
- Mengetahui spesifisitas substrat
- Mengetahui efek inhibitor enzim ( kecepatan
reaksi katalitik)
 Vo (Initial velocity )
• One simplifying approach in kinetics experiments is
to measure the initial rate (or initial velocity),
designated V0, when [S] is much greater than the
concentration of enzyme, [E
• When enzyme is mixed with high concentration of
substrate [S] reaction goes rapidly to steady state.
• Use low starting [S] and increase
• Hold [enzyme] constant
• Measure initial rate of reaction, Vo as [S] increases
– Until rate becomes constant: approaches Vmax
Effect of [substrate] on RX Velocity
Michaelis-Menten Equation

Vmax [S]
V0 =
Km +[S]
Effect of Substrate Concentration on
Reaction Velocity
 k1 k2
E+S ES E+P
k-1 k-2
Step 1. Formation of the ES complex:
• formation an enzyme-substrate complex in a
relatively fast reversible step:
• forward rate v1 = k1[S][E]
• backward rate v-1 = k-1[ES]
• K = k1/k-1
• k1 k2
E+S ES E+P
k-1 k-2

• Step 2. Formation of products:

• ES complex breaks down in a slower step to yield
the free enzyme and the P  limit the reaction
• forward rate v2 = k2[ES]
• backward rate v-2 = k-2[E][P]
• Breakdown of ES to form products is assumed to
be slower than formation of ES (v1) or breakdown
of ES to re-form E and S (v-1)
• So the initial rate v0 = k2[ES]
• Vo easily measure initial rates, but [ES] is very
difficult to measure
• The mathematical equation
• relationship between the rate of an enzyme
reaction and the substrate concentration is the
Michaelis-Menten equation:
Vmax [S]
V₀ = -------------
Km + [S]
V₀ is the observed velocity at the given [S]
Km is the Michaelis-Menten constant
Km = (K-1 + K2) / K1
Vmax is the maximum velocity at saturating [S] conc.
Lineweaver-Burk Plot
1 Km 1 1
 
v Vmax[S] Vmax
 Reversible inhibitors can be
classified into :

• Competitive

• un-competitive

• mixed/non-competitive
Competitive Inhibition
 KI 
E I
EI
Vmax S
vo 
K M  S


  1 
I 

 KI 
Competitive Inhibition: Lineweaver-Burke Plot
Uncompetitive Inhibition
Uncompetitive Inhibition
Uncompetitive Inhibition: Lineweaver-Burke Plot
Mixed inhibition
Mixed inhibition
 Mixed inhibition is when the inhibitor binds to the
enzyme at a location distinct from the substrate
binding site. The binding of the inhibitor will either
alter the KM or Vmax or both.

E I KI 
ESI
KI 
EI ESI
Vmax S 
   1 
I 

vo 
K M   S  K 
I 