Solutions to exercise 4 in TBT4102 Biochemistry 1

1) For any reaction the rate is determined by the reaction constant and the concentration of
substrate(s). The reaction constant is dependent on the activation energy and the higher the
activation energy is, the slower the rate is. The activation energy is the difference in free
energy between the ground state and the transition state. Enzymes work by lowering the
activation energy for the reaction and thus making the reaction go faster.

The binding energy (ΔGB) is the difference between the activation energy for the uncatalyzed
reaction (ΔG╪uncat) and the activation energy for the catalyzed reaction (ΔG╪cat). This energy
comes from weak interactions between the substrate and the enzyme. These interactions result
in release of small amounts of energy (=binding energy).

2) The Michaelis-Menten equation describes the relationship between the initial rate and the
substrate concentration: V0 = (VMAX[S])/(KM + [S])
The initial rate increases as the concentration of S is increased. At very low concetrations of S
the initial rate will exhibit a linear dependence on S (KM>>[S], equation simplifies to V0 =
(VMAX[S])/KM). However, at very high concentrations of S the initial rate will reach a plateau.
This plateau represents the VMAX. ([S]> >KM, equation simplifies to to V0 = VMAX).

b) Invert the Michaelis-Menten equation and write it on the form y=ax + b, where
y = 1/V0 og x = 1/[S].

The Lineweaver-Burk plot will give a straight line where the intercept with the y-axis
represents 1/VMAX, the intercept with the x-axis represents -1/KM and the slope represents
KM/VMAX. Thus it can be used to calculate the KM and VMAX values. The Lineweaver-Burk
plot is easier to analyze because the equation is of the form y=ax + b, giving a straight line.

c) First calculate the 1/[S] and 1/[V] values:

1/[S] μM-1 1/V min/μmol
0,17 0,044
0,10 0,031
0,05 0,021
0,02 0,014
0,01 0,012

Plotting these values should give a straight line:

0,050
y = 0,1996x + 0,0108
0,040

0,030

0,020
1/V

0,010

0,000
-0,15 -0,10 -0,05 0,00 0,05 0,10 0,15 0,20
-0,010

-0,020

1/[S]

Intercept with y-axis gives the 1/VMAX= 0,0108→VMAX= 93 μmol/min.

Slope gives KM/VMAX=0,1996→KM= 19 μM (1,9×10-5 M)

d) Calculate the 1/VI values as well:

1/[S] 1/V 1/VI

0,17 0,044 0,083
0,10 0,031 0,070
0,05 0,021 0,060
0,02 0,014 0,053
0,01 0,012 0,051
 0,100

0,080

0,060
1/V

0,040
No inhibitor
With inhibitor
0,020

0,000
-0,40 -0,30 -0,20 -0,10 0,00 0,10 0,20

-0,020

1/[S]

The plot results in parallell lines, meaning that the intercepts with the x-and y-axis are
affected by the addition of the inhibitor, but not the slope. This means that the inhibitor is
uncompetitive (1/V0=(KM/VMAX)×(1/[S]) + α’/VMAX→slope not affected)

3) Calculate the 1/[S] and 1/[V] values:

1/[S] 1/V 1/VA 1/VB

0,8 0,58 0,97 0,99
0,60 0,49 0,79 0,79
0,4 0,38 0,61 0,58
0,2 0,30 0,43 0,39
0,1 0,24 0,34 0,29

Plot the three data sets and compare:

1,2

0,8

0,6
No inhibitor
1/V

0,4 Inhibitor A
Inhibitor B
0,2

0
-0,6 -0,4 -0,2 0 0,2 0,4 0,6 0,8 1
-0,2

-0,4
1/[S]

a) Inhibitor A gives mixed inhibition as both slope and intercepts with x-and y-axis is
affected.
b) Inhibitor B gives competitive inhibition as the slope and intercept with the x-axis is
affected, but not the intercept with the y-axis.
4) a)Enantiomers: stereoisomers that are nonsuperposable mirror images of each other.
b) Epimers: two sugars that differ only in the configuration around one carbon atom.
c) Anomeric carbon atom: the hemiacetal/hemiketal carbon atom (C-1).
d) Anomers: isomeric forms of monosaccharides that differ only in their configuration about
the hemiacetal or hemiketal carbon atom.

5) a)

Glucose is an aldose because the carbonyl group is at the end of the carbon chain (i.e. an
aldehyde group)

b)

There will be two forms of glucose present in aqueous solution, the α and β anomers. The
formation of the ring structure produce an additional asymmetric (chiral) carbon atom (C-1)
and it will therefore exist as two stereoisomeric forms.

c) The cyclic (ring) form of glucose is called glucopyranose, because compounds with six-
membered rings are called pyranoses (resemble pyran).