BC2014 Problem set 6 Enzymes and kinetics

1. The following data were obtained from an enzyme kinetics e!eriment.

"ra!h the data #sing a $inewea%er&B#rk !lot and determine' by ins!ection of
the gra!h' the %al#es for (m and )ma.
*+, -./0 ) -nmol1min0
2222222 22222222222
0.20 1.43
0.26 1.64
0.33 2.05
1.0 3.33
2. Calc#late the maim#m rate for an enzyme if its kcat 6 1.4 10
4
s
&1
(m 6 70
./. 8s this enzyme %ery efficient9
3. :se the /ichaelis&/enton ;<#ation to calc#late the missing %al#es of *+,
gi%en below if )ma 6 = mmol1min. Plot *+, %ers#s ) ->?T the reci!rocals@0.
Araw line !arallel to the &ais at )ma and etend yo#r !lotted line to show its
a!!roach to )ma.
*+, -m/0 ) -mmol1min0
2222222 22222222222
10 1.2
9 1.4
9 2.1
9 2.2
9 2.=
4. The effect of an inhibitor on an enzyme was tested and the e!eriment ga%e
the res#lts below. Plot the data and determine' by ins!ection of the gra!h'
what ty!e of inhibition is in%ol%ed.
*+, ./ ) -.mol1min0 ) -.mol1min0 ) -.mol1min0
with 0.0 n/ with 2= n/ with =0 n/
8nhibitor 8nhibitor 8nhibitor
222222 22222222222 22222222222 22222222222
0.4 0.22 0.21 0.20
0.64 0.27 0.26 0.24
1.00 0.32 0.30 0.25
2.00 0.40 0.36 0.32
5.. You have purified a new enzyme and want to determine its
kinetic properties in the presence and absence of two inhibitors (I-1
and I-2). These are the experimental conditions:
1ml reaction volume, pH 7.5, 0.1 M KCl
the substrate will be varied between 1 and 20 mM
amount of enzyme added = 0.2 microgram (molecular weight
determined by SDS gel analysis= 100,000 g/mol)
co-substrates are required, but are added in excess
incubation time, 5 min at 25C
Under these conditions the following data were obtained:

Velocity, nanomol/min
[S], mM no inhibitor 5 mM I-1 10 mM I-2
1 2.5 1.17 0.77
2 4 2.1 1.25
5 6.3 4 2
10 7.6 5.7 2.5
20 9 7.2 2.86
A.. Plot v vs [S] on graph paper and estimate the Km and Vmax for
each set of data. What type of competitors are I-1 and I-2?
B. Plot 1/v and 1/[S] on graph paper and determine the Km and
Vmax for each set of data. How do they compare with the answers
in B?
C. From the Vmax (uninhibited enzyme), can you estimate the
turnover number in molecules/sec?

Problems from /atthews tet Cha!ter 11.
=.
13.
14. :se e<n 11.5 to answer !art -d0.
14. The ;adie&Bofstee !lot.