BBCCL-108 ENZYMES

EXPERIMENT 4
DETERMINATION OF Km
AND Vmax USING
LINEWEAVER – BURK
GRAPH

Structure
4.1 Introduction 4.5 Determination of Km and
Vmax Using Lineweaver –
Expected Learning Outcomes
Burk plot/graph - Protocol
4.2 Principle
4.6 Precautions
4.3 Reagents
4.7 Summary
4.4 Preparation of Enzyme
Extract

4.1 INTRODUCTION
You have learned in core course BBCCT-107 of Enzymes that various factors
e.g., substrate availability, pH of medium, temperature, presence of
activator/inhibitor etc. can affect the rate of an enzyme catalyzed reaction.
Among these, substrate concentration is very important factor affecting the
rate of reaction. E and S combine to form [ES] complex which is converted to
[EP] from which finally the product P is released. With the increase in
substrate concentration, velocity of reaction is increased in a hyperbolic
manner (See Fig. 4.1). At low substrate concentration, not all the active sites
of enzyme are occupied by substrate molecules and the rate of reaction will be
proportional to the substrate concentration exhibiting first order reaction
kinetics. When the substrate concentration is very high, enzyme is saturated
i.e., the number of substrate molecules exceeds the number of active sites of
the enzyme. Under this condition, increase in substrate concentration will not
affect the velocity of enzyme catalyzed reaction i.e., velocity will be
independent of the substrate concentration. At this stage velocity is at a
maximum (Vmax), and zero order kinetics is observed.

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Experiment 4 Determination of Km and Vmax using
Lineweaver-Burk Graph

Fig. 4.1: Effect of substrate concentration on enzyme activity.

The mathematical expression of the hyperbolic shape curve

curve is given by
Michaelis-Menten equation:

Vmax [ S ]
V0 
Km  [S ]

It shows relationship between initial velocity (Vo ) with maximum velocity

(Vmax), initial substrate concentration [S] and the Michaelis constant ( Km ).

In this equation, if [ S] = Km, the Vo= Vmax/2

Therefore, Michaelis constant (Km) can be defined as the substrate

concentration at which the initial velocity is half of the maximum velocity. It is
expressed in the units of substrate (M or mM).

Km is a measure of affinity of an enzyme for its substrate. A large Km means

low affinity while a low Km value indicates high affinity for the given substrate.

Vo vs. [S] curve gives only an approx. value of Km as Vmax is not clearly
determined.

Lineweaver-Burk plot is obtained by transformation of Michaelis-Menten

equation and is a very common method to determine Km and Vmax.

Taking reciprocal on both sides of equation;

1 Km 1 1
 . 
V0 Vmax [ S ] Vmax

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BBCCL-108 ENZYMES

It is a straight line equation. By plotting a graph 1/Vo vs. 1/[S], Vmax on Y-axis
and Km from X-axis can be obtained. It is also called double reciprocal plot as
1/V0 vs. 1/[S] are plotted (Fig. 4.2).

Fig. 4.2: Lineweaver-Burk plot (Double Reciprocal plot).

Expected Learning Outcomes

After studying this unit, you should be able to:

 understand how substrate level affects the rate of an enzyme catalyzed

reaction; and

 perform the experiment to study the effect of substrate concentration on

activity of acid phosphatase and determine Km and Vmax using
Lineweaver-Burk plot/graph.

4.2 PRINCIPLE
The hyperbolic curve (initial velocity V0 vs. substrate concentration) is not
entirely satisfactory for determination of Km and Vmax for an enzyme catalyzed
reaction. The Michaelis-Menten equation can be rearranged by taking
reciprocals on both sides of equation to a form that is equivalent to the
straight-line equation. By plotting 1/Vo on Y-axis (ordinate) and 1/[S] on X-
axis (abscissa), a straight line relationship is observed which is known as
Lineweaver- Burk plot, where the slope is Km/Vmax and the intercept on the Y-
axis is I/Vmax. The X-intercept is -1/Km where Km is the Michaelis-Menten
constant. In acid phosphatase catalyzed reaction using p-nitrophenyl
phosphate as substrate, the inverse values of different concentrations of p-
nitrophenol (i.e., 1/Vo) vs. the inverse values of pNPP concentrations (i.e., 21
Experiment 4 Determination of K m and Vmax using
Lineweaver-Burk Graph

1/[S]) are plotted to determine the Km and Vmax from the Lineweaver and Burk
plot.
Note:

1. Only approximate values of Vmax and Km can be obtained from velocity

(Vo) vs. [S] graph.

2. Reactions for substrate conversion etc. are given in Expt. 1.

4.3 REAGENTS
i) Buffer: 0.1M Sodium citrate buffer (pH 4.8) as described in experiment
1.

ii) Substrate solution: 5 mM p-nitrophenyl phosphate (pNPP), dissolved in

0.1 M sodium citrate buffer (pH 4.8).

iii) 0.2N Sodium hydroxide solution

4.4 PREPARATION OF ENZYME EXTRACT

Follow the steps as already explained in Ex. 1.

4.5 DETERMINATION OF Km AND Vmax USING

LINEWEAVER-BURK PLOT/GRAPH-
PROTOCOL
Take clean test tubes numbered from 1 to 9. Pipette out different volumes of 5
mM pNPP solution and the citrate buffer as follows (in duplicate). Prepare
another set also and mark as Blank e.g. B1, B2, B3, B4 B5, B6, B7, B8, B9.

Reagent Tube

1 2 3 4 5 6 7 8 9

5 mM pNPP 0.025 0.05 0.10 0.15 0.20 0.25 0.3 0.6 0.9
solution (mL)

0.1M Citrate buffer 0.875 0.85 0.80 0.75 0.70 0.65 0.6 0.3 -
(pH 4.8) (mL)

o
Mix properly and equilibrate at 37 C for ~3 min

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BBCCL-108 ENZYMES

 Start the reaction by adding 0.1 mL of enzyme extract in each tube (assay
tubes only). Note down the time of adding the enzyme extract in first tube.
o
 Immediately mix properly and incubate at 37 C for 10 min.

 Stop the reaction by adding 4.0 mL of 0.2N NaOH in each tube.

 In blanks, add enzyme extract (0.1 mL in each tube) at the end after
addition of 0.2N NaOH.

 Record the absorbance of each tube (assay) against the corresponding

blank (prepared for each pNPP concentration), at 410 nm using a
spectrophotometer.

Calculations and plotting the graph

 Determine the amount of p-nitrophenol produced in each tube from the

standard curve prepared as explained in experiment 1.

 Calculate the µmoles of p-nitrophenol produced/min (termed as velocity,

V0) for each concentration (mM) of pNPP, represented as [S].

 Plot V0 vs. [S] and note the shape of curve.

 For the Lineweaver-Burk plot (double reciprocal plot), you will have to find
out the reciprocal of velocity (1/V0) and substrate concentration (1/[S]).

Plot 1/ V0 on Y-axis and 1/[S] on X-axis, and determine the value of Km

and Vmax of the acid phosphatase catalyzed reaction.

Note: i) Prepare blank tube corresponding to each substrate concentration.

ii) The velocity of enzyme catalyzed reaction is expressed as product

formed per unit time e.g. µmoles p-nitrophenol/min.

4.6 PRECAUTIONS
i) The substrate concentration should cover a wide range.

ii) Pipetting of volumes should be very accurate.

iii) Always wear gloves while working with p-nitrophenylphosphate and p-

nitrophenol, avoid contact, inhalation etc.

iv) Ensure equilibration at incubation temperature before adding the enzyme

extract.

4.7 SUMMARY
In an enzyme catalyzed reaction as the substrate concentration is increased,
the velocity of reaction increases till it reaches the maximum value (Vmax). The
substrate concentration at which the velocity of enzyme catalyzed reaction is
half maximal is defined as the Michaelis constant (Km). Km is a measure of
affinity of the enzyme for its substrate and its units are same as that of
substrate. A double reciprocal plot (1/Vo vs. 1/[S]), known as Lineweaver-Burk 23
Experiment 4 Determination of K m and Vmax using
Lineweaver-Burk Graph

plot, is a common method to determine the Km and Vmax. Activity of acid

phosphatase can be measured by using a chromogenic synthetic substrate, p-
nitrophenylphosphate. The velocity is calculated as µmoles p-nitrophenol
formed/min and Km is expressed as mM. For an enzyme assay, the reaction is
carried out under optimal conditions of pH, temperature, substrate
concentration etc. To set up the enzyme assays, substrate should not be
limiting i.e., enzyme should be saturated with the substrate.

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