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ENZYMES Problems and Solutions

The document provides data from several enzymatic reactions measured at different substrate concentrations. It then explains how to analyze the data using Lineweaver-Burk plots to determine the Michaelis-Menten constants, KM and Vmax. Specifically: 1) The data is converted to reciprocals of substrate concentration and velocity before plotting to obtain linear relationships. 2) The y-intercept of the Lineweaver-Burk plot equals 1/Vmax and the x-intercept equals -1/KM. 3) Examples are shown for competitive, noncompetitive, mixed, and uncompetitive inhibition based on how KM and Vmax are affected.

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1K views6 pages

ENZYMES Problems and Solutions

The document provides data from several enzymatic reactions measured at different substrate concentrations. It then explains how to analyze the data using Lineweaver-Burk plots to determine the Michaelis-Menten constants, KM and Vmax. Specifically: 1) The data is converted to reciprocals of substrate concentration and velocity before plotting to obtain linear relationships. 2) The y-intercept of the Lineweaver-Burk plot equals 1/Vmax and the x-intercept equals -1/KM. 3) Examples are shown for competitive, noncompetitive, mixed, and uncompetitive inhibition based on how KM and Vmax are affected.

Uploaded by

Chadby GraNaNo
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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The following data describe an enzymed-catalyzed

reaction. Plot these results using the Lineweaver-Burk


method, and determine values for KM and Vmax.

Substrate Velocity
Concentration (mM/sec)
(mM)
2.5 0.024
5 0.036
10.0 0.053
15 0.060
20 0.061
Since the given data are the [S] and velocity, you need to compute for
their reciprocals before you can use the Line weaver Burk equation.

[S] 1/[S] V 1/V


(mM) mM -1 (mM/sec)
2.5 0.4 0.024 41.67

5 0.2 0.036 27.78

10 0.1 0.053 18.87

15 0.06 0.060 16.67

You can compute for the KM and V max in 2 ways:


•Plotting the points (x axis would be the 1/[S] while the y axis 1/V). Get the y
intercept, that is the reciprocal of V max while the negative x intercept is the-1/ KM
(since the question asked you to plot, this should be yourway).
•Or using the linear regression formula of your calculator, compute for the slope
(m) and the y intercept (b)
• Answer: b=12.07, Vmax = 0.08 mM/sec (b=1/Vmax , so 12.07=1/Vmax)
m= 74.61, Km= 5.97 mM (m=Km/Vmax so 74.61= Km/0.08))
The velocity of an enzyme-catalyzed reaction was
measured at several substrate concentrations.
Calculate Km and Vmax for the reaction

1/[S] (M-1) 1/Vo (mM-1.s)


0.25 0.75
0.5 1.20
1.0 1.71
2.0 2.18
4.0 2.53

The values are already in their reciprocal, so you can use


the data as they are.
Answer:
b=1.007, Vmax=0.99mM/sec
m= 0.43 Km=0.42 μM
The figure below shows the Lineweaver–Burk plots for the two sets of data
of an enzyme catalyzed reactions in the presence and absence of an
inhibitor.
1. What is the Km and Vmax of
Reaction A? Reaction B?
Reaction A
A b= 20 Vmax=0.05 Au/min
-x intercept= -0.8 KM=1.25 mM
Reaction B
B b=10 Vmax=0.1AU/min
same Km
2. Which is the inhibited reaction?
Reaction B is the inhibited reaction,
non-competitive (lower Vmax,
same Km)

NOTE; b and –x intercept values are


based on the plots
Sucrose is hydrolyzed to glucose and fructose in a classic experiment in
kinetics. The reaction is catalyzed by the enzyme invertase. Using the
following data, determine, by Lineweaver-Burk method, whether the inhibition
of this reaction by 2 M urea is competitive or noncompetitive

Sucrose 1/[S] V, No I 1/V, No I V, With I 1/V, with I


concentrati (arbitrary (arbitrary
on (M) unit) unit)
0.0292 34.24 0.182 5.49 0.083 12.04

0.0584 17.12 0.265 3.77 0.119 8.40

0.0876 11.41 0.311 3.21 0.154 6.49

0.117 8.54 0.330 3.03 0.167 5.98

0.175 5.71 0.372 2.69 0.192 5.21

b =2.14 Vmax=0.47 b=3.92 Vmax=0.26

m=0.10 Km=0.05M m=0.24 Km=0.06 M


Lower Vmax, Higher Km, The inhibition is mixed
uncompetitive
The kinetics of an enzyme are measured as a function
of [S] in the presence and absence of 2 mM I. Compute
Km and Vmax in the absence and presence of I
Without I With I
[S] 1/[S]
(uM) V 1/V V 1/V
(uM/min)

3 0.33 10.4 0.10 4.1 0.24


5 0.20 14.5 0.07 6.4 0.16
10 0.10 22.5 0.04 11.3 0.09
30 0.03 33.8 0.03 22.6 0.04
90 0.01 40.5 0.02 33.8 0.03
b=0.02 Vmax=50 uM/min b=0.02 Vmax=50 uM/min
m=0.25 Km=12.5uM m=0.67 Km=33.5μM

Same Vmax, higher Km, Competitive

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