SU 3

Kinetics of single substrate enzyme

catalysed reactions
 Michaelis- Menten
Henri (1903) and Michaelis and Menten (1913) made the
following assumptions for single substrate reactions with
single binding site enzymes:
• At to [P] can be ignored as well as k-2
• Immediate equilibrium between E, S en ES – product
production not a factor
• [E] = [Eo] - [ES]
• vo = k2[ES]
• At ↑[S] is [Eo] = [ES], then vo = Vmax or Vmax = k2[Eo]
• [So] >>> [Eo], thus at to [S] = [So]

Which is to say: vo = (Vmax[So])/([So] + Ks)

Know derivision!! (p106-107)
Video link:
https://www.youtube.com/watch?v=8PWF5OeB7Ec
 Briggs-Haldane modification
• [ES] <<< [S], [ES] <<< [Eo]
• [ES] stays constant, while [S] and [P] changes (steady-
state)
• Thus formation rate of ES = breakdown rate of ES
• Km = (k-1 +k2)/k1 = ([E][S])/[ES]
• Rest of assumption as M-M

– vo = (Vmax[So])/([So] + Km) = M-M equation

Know derivision!! (p108-109)

Video link:
https://www.youtube.com/watch?v=YSOJNKQeCtQ
 Meaning of M-M equation
• For which reactions does it count? NB!
– Single substrate, one S-binding site per enzyme
– Single ES (I)
– ES → EP rate limiting step
• Kcat = turnover number
• Vmax = maximum reaction rate at set
conditions
• Km vs Ks … what’s the difference and
meaning?
• Kcat/Km = Catalytic efficiency
• See example of phosphofructokinase and
triosephosphate isomerase NB!!
 Lineweaver-Burk plot
Video link: https://www.youtube.com/watch?v=4aVOtcpGaZc
Eadie-Hofstee graph (L-B x
Hanes graph (L-B x [So])
vo.Vmax)
https://www.youtube.com/watch?v=n_
https://www.youtube.com/watch?v= 3AkzEzLEY
_qSJFBoxdwQ
 Deviations from stationary state
kinetic assumptions
• Assumption Critisism
• At to, [P] and k2 is negligible ↓ Haldane relationship discusses this
deviation
• Immediate equilibrium between B-H says [ES] stationary while [S] [E],
[S], [ES] and [P] change
• [Eo] = [E] + [ES] and Enzyme unstable in vitro, thus
• MM- eq true if [Eo] is constant [Eo] = [E] + [ES] + [Ed]
• Vo = k2[ES] and [P] and –k2 negligible [P] en –k2 not always negligible
• ES → E + P rate limiting step ES → E + P can be more complex
thus k2 not constant for rate limiting step
• At ↑[S] is [Eo] = [ES] and Vo = Vmax or This step can be blocked and is then
Vmax = k2[ES] incompatable with the law of mass
action

6. At to is [S] = [So] or [So] >>>>[Eo] Not “assumption”, but “approximate”

Theoretical progress curve
 Relaxation kinetics
[Δ A] = [Δ A]₀ e –t/τ
1/τ = k1([E ] + [S ]) + k-1
eq eq
• Kcat = Vmax/[Eo]

• Vmax = 12 µmol/min of 12 x 10-6 mol/min

• [E] = 10 ng met Mw = 60 000 g/mol

• = 10 x 10-9g/60 000g/mol
• = 1.667 x 10-13 mol

• thus

• Kcat = 12 x 10-6 mol/min/1.667 x 10-13 mol

• = 72 000 000 min-1 of 7.2 x 107 min-1
Introduction to enzyme kinetics:
https://www.youtube.com/watch?v=kmyR1
cYxRL4