ENZYMES 1

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ENZYMES 1 Phil Rowe All presentations available
at www.staff.livjm.ac.uk/phaprowe/ (Select
Proteins and enzymes)
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Part 1 Enzymes as biological catalysts
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Spontaneous reaction
A B ( Energy released)
Spontaneous, but may actually occur very
slowly or not at all.
A
Energy IN
Energy OUT
A
B
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Enzymes Biological catalysts
Enzymes provide an alternative route of reaction
i
ii
iii
iv
A E A.E A.E B.E B E
Energy IN
Energy OUT
Energy is required for the activation of A.E to
A.E, but not as much as for A to A
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Progress of reaction with or without enzyme
catalysis
A
Energy
No enzyme With enzyme
A
Net energy release
B
Progress of reaction
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Proportion of molecules possessing enough energy
to become activated
ProportIon
Energy
A.E A.E
A A
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Net energy release and equilibrium constant
Many biochemical reactions have to be considered
as reversible
A B
At equilibrium, a certain proportion of the A
will have been converted to B. But in the case
of reversible reactions the process may stop
short of completion.
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Net energy release and equilibrium constant
The equilibrium position is linked to free energy
release. The more energy that is released, the
more complete the reaction will be. We saw
previously (Slide 5) that net energy release is
exactly the same with or without an enzyme. It
therefore follows that the involvement of an
enzyme will not change the final equilibrium
position.
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Effects of enzymes

• Reaction rate increased
• Final equilibrium position unchanged

We end up at the same place but get there quicker.
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Enzyme, active site, substrate and product
Substrate
Product
Enzyme
Active site
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Enzyme structure
Enzymes generally consist of a protein molecule
that is folded up so as to leave a deep cleft on
the surface into which the substrate fits - the
active site
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Part 2 Enzymes kinetics
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Enzyme kinetics
Study of the rate of enzyme catalysed reactions.
Particularly

• E constant
• S variable
• See effect on velocity (v)

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Low substrate concentration
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Higher substrate concentration
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Even higher substrate concentration
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Very high substrate concentration
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Velocity (v) versus substrate concentration
Velocity (v)
(microMole/min)
All active sites on enzyme saturated with
substrate

Substrate concentration (microM)
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Part 3 Vmax and Km
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Vmax
Vmax 10microMole/min
Velocity (v)
(microMole/min)
All active sites on enzyme saturated with
substrate

Substrate concentration (microM)
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Vmax
The velocity of reaction at infinite substrate
concentration. A measure of the capacity of the
enzymes active sites.
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Differing affinities
High affinity
Velocity (v)
(microMole/min)
Low affinity

Substrate concentration (microM)
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Km
Vmax
Vmax/2
Velocity (v)
(microMole/min)

Substrate concentration (microM)
Km1
Km2
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Km
The concentration of substrate that will 50
saturate the active sites of an enzyme (Cause
50 of Vmax). Enzyme 1 has a higher affinity than
2, but a lower Km value. Km is an inverse
measure of affinity. (The lower the Km, the
higher the affinity.) Measured in concentration
units.
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Part 4 The Michaelis-Menten equation
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The Michaelis-Menten equation
The Michaelis-Menten equation allows us to
calculate the velocity (v) of an enzyme catalysed
reaction, from the substrate concentration (S)
and the two kinetic parameters (Vmax and Km).
v Vmax . S
Km S
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The Michaelis-Menten equation
If the relevant values
are S 20microM
Vmax 15microMol/min
Km 40microM v Vmax . S
Km S 15microMol/min x 20microM
20microM 40microM 15microMol/min x
20microM 60microM
5microMol/min
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The Michaelis-Menten equation
Note You would not be expected to learn the
Michaelis-Menten equation, but you should be
aware of its existence and its role in allowing
the calculation of the velocity of an enzyme
catalysed reaction.
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Part 5 Analysing data from an enzyme kinetics
experiment
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Extrapolate curve to get Vmax ???
10 5 0
Velocity (v)
(microMole/min)
0 50
100
Substrate concentration (microM)
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Linearise the dataThe Lineweaver-Burk plot
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1/v
1
-1/Km
1/Vmax
-1,000 0 1000
2,000 3,000
1/S
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Prepare data for the Lineweaver-Burk plot
S v 1/S
1/v(microM) (microMole
/min)
5 15 25 50100
1.73.85.06.78.0
0.20.0670.040.020.01
0.590.260.200.150.125
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Use Lineweaver-Burk plot
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4
1/v
-1/Km -0.04
2
1/Vmax 0.105
0
0.1
0.2
1/S
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Results from Lineweaver-Burk plot
1/Vmax 0.105Vmax 9.52microMole/min -1/Km
-0.041/Km 0.04Km 25microM
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Terms with which you should be familiar
Active site Substrate Product Enzyme
kinetics Velocity (v)
Vmax Km Michaelis-Menten equation Lineweaver-Burk
plot
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What you should be able to do

• Describe the effect of an enzyme on the rate and
  final equilibrium of a chemical reaction
• Describe the mechanism by which enzymes increase
  the rate of a reaction
• Describe the binding of a substrate to the
  active site
• Describe the relationship between substrate
  concentration and velocity

Continued on next slide
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What you should be able to do

• Describe the information conveyed by Vmax and Km
• Know that v can be related to substrate
  concentration, Vmax and Km via the
  Michaelis-Menten equation.
• Present enzyme kinetics data as a
  Lineweaver-Burk plot and obtain Vmax and Km

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Appendix Linearisation using the Lineweaver-Burk
Plot
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Linearisation of the data
Start with the Michaelis-Menten equation v Vmax
. S Km S 1 Km S v Vmax .
S 1 Km Sv Vmax.S
Vmax.S 1 Km 1v Vmax.S
Vmax
Invert
Open up into 2 terms
Cancel S
Continues
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Gradient Km/Vmax
1 Km 1v Vmax.S Vmax 1
Km x 1 1v Vmax S Vmax
1/v
Intercept 1/Vmax
1/S
Gradient a
Y a x X c
Y
Intercept c
X
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Obtaining Km
Can theoretically obtain Km from the gradient of
the graph (Km/Vmax), but inconvenient in
practice. Instead, extrapolate the graph to the
left until it meets the horizontal axis. Consider
the triangle formed-Height hBase
bGradient of the hypotenuse h/b Gradient
h/bb h/gradientb (1/Vmax) / (Km/Vmax)b
1/Km As we are extrapolating to the left, the
intercept on the horizontal axis is at 1/Km.
1/v
Gradient Km/Vmax
-1/Km
h 1/Vmax
0
b
1/S
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Obtaining Vmax and Kmfrom the Lineweaver-Burk
plot
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1/v
1
-1/Km
1/Vmax
-1,000 0 1000
2,000 3,000
1/S