BCM-262

Prof. DeRidder

Purification and Citrate Inhibition of Fumarase from Yeast

Nathan Zaroban 18, Sydney Vrecenar 18, Zach McGriff 17

Fumarase is an enzyme that catalyzes both the hydration of fumarate

and the dehydration of L-malate, and is a key component of the citric
acid cycle. Since all aerobic organisms utilize this cycle to generate
energy, studying the kinetic activity of the enzyme proves to be quite
beneficial. We purified a recombinant version of fumarase from
Saccharomyces cerevisiae, better known as Bakers yeast, using
immobilized metal affinity chromatography (IMAC) with a
nitriloacetic acid (NTA) agarose column 1 This particular enzyme
contains a histidine tag that actively binds to nickel, making this form
of purification possible. At each stage of purification, fumarase
specific activities were calculated. Michaelis-Menton assays were
performed for both the forward and reverse reactions using the
purified fumarase samples, and class data was pooled to calculate
kinetic parameters.

Figure 1. SDS/PAGE Purity Analysis. A= MW Standards,

B= fumarase standard, C= lysate, D= flow-through, E= 5%
buffer B wash, F=dialyzed fumarase, X= nothing. Both the
fumarase standard and our dialyzed fumarase sample bands
are near 45 Kd. The single relatively strong band and few
very faint bands in F indicates that the dialyzed fumarase
sample was very pure.
45 Kd-

Sample Activity Calculations

Fumarase Activity (Units/mL)=
(a(250 nm))/(1450 M-1cm-1) X (0.003 L) X (106 mol/mol)
X Dilution Factor/ 0.03 mL

Figure 4. Michaelis-Menten plot of fumarasecatalyzed conversion of L-malate to fumarate in

the presence of varying concentrations of citrate.
Each reaction was ran twice and the initial velocities
were averaged.
Group
Km (mM) Vmax
(mol/min)

Protein Concentration (mg/mL)=

calculated from Bradford Assay standard curve
Specific Activity (Units/mg protein)=
Fumarase Activity (Units/mL)/Protein Concentration (mg/mL)
Purification
Sample

Our independent project investigated the inhibitory effects of citrate

(prevalent in the citric acid cycle, as the name implies) on the
enzymatic capabilities of fumarase. Previous studies have shown that
citrate acts as a competitive inhibitor of fumarase2,3,4 We set out to test
for ourselves whether citrate would act as a competitive or noncompetitive inhibitor, and hypothesized that citrate would act as a
competitive inhibitor2,3.4. To determine this, Michaelis-Menton assays
were performed on the fumarase-catalyzed dehydration of L-malate in
the presence of varying concentrations of citrate.

Citrate Inhibition

Results: Purification/Activity Assay

Introduction

Lysate
Dialyzed
Fractions

Volume
(mL)

Fumarase Activity
(units/mL)

0.85
2.85

Total Fumarase [Protein]

Activity (units) (mg/mL)

34.55

29.37

70.30

Total Protein
(mg)

33.20

199.5

0.344

Specific Activity
(Units/mg
protein)

28.22
0.904

Fold Purification

1.040
204.4

Figure 2. Michaelis-Menten plot of fumarase catalyzed

conversion of fumarate to L-malate. Two reactions were
ran at each concentration of fumarate and the initial
velocities were averaged.

Sample Calculations
Km (mM) = (-1/x-intercept of Lineweaver-Burk)
Vmax (mol/min) = (1/y-intercept of Lineweaver-Burk)
Kcat (conversions/min) = Vmax/ Total [Fumarase] (mol)

Data Group

Vmax
(mol/min)

Kcat
(conversions/
min)

Our DataFumarate

1.33

1.68

8927

Class AverageFumarate

1.36

2.06

8613

Class AverageMalate

6.65

0.485

1133

Total [Fumarase] (mol)=

[Fumarase from Bradford] (mg/mL) X 0.03 mL X (1g/103 mg)
X (1 mol/ 212,980 g) X 4 active sites X (106 mol/mol)
Keq= (Kcat (fum) X Km (mal))/ (Kcat (mal) X Km (fum))

Km (mM)

Table 2. Kinetic measurements of both fumarasecatalyzed reactions using purified/dialyzed fumarase

samples. Keq= 37.12

6.25 mM Citrate

6.046

0.7794

18.5 mM Citrate

9.539

0.9734

KI (18.5 mM Citrate)= 8.11 x 10-3

Discussion/Conclusions

Table 1. Calculation of enzyme activity, concentration, and specific activity for all aliquots collected demonstrating
fumarase activity. Aliquots not showing activity were excluded. Fumarase activity was measured by conversion of lmalate to fumarate. Units= mol of fumarate produced/min. Protein concentration was determined by the Bradford assay.

Figure 3. Lineweaver-Burk plot of fumarase-catalyzed

conversion of fumarate to L-malate.

1.024

KI (6.25 mM Citrate)= 5.79 x 10-3

196.5x

Methods

2.908

Table 3. Km and Vmax for the

uninhibited fumarase-catalyzed
conversion of L-malate to fumarate, and
the same reaction in the presence of 6.25
mM and 18.5 mM Citrate.

KI = Dissociation Rate of Fumarase and Citrate =(Km x [I])/(Kmapp-Km)

1x

Kinetics

Uninhibited Control

Figure 5. Lineweaver-Burk plot of fumarasecatalyzed conversion of L-malate to fumarate in

the presence of varying concentrations of
citrate.

SDS/PAGE analysis shows that the dialyzed fumarase sample (approx. 45 Kd) is very pure.
The massive increase in specific fumarase activity from the lysate sample to the dialyzed
fumarase also demonstrates the high purity of the fumarase sample (196.5x purification).
Our Km, Vmax and Kcat measurements were close to that of the other fumarate group in our
lab. The calculated Keq for the forward and backward reactions was 37.12. One of the malate
groups had a Km that was approximately 10-fold larger than the other group. We must take
this into consideration when interpreting this data.
Both 6.25 mM Citrate and 18.5 mM Citrate successfully inhibited fumarase activity.
The Vmax of the reactions in the presence both citrate concentrations is fairly close to the
Vmax of the uninhibited reaction. This loosely supports previous studies and our hypothesis
that citrate acts as a competitive inhibitor 2,3,4. However, this experiment should be repeated
with greater sample sizes, at greater concentrations of L-malate, with smaller intervals of Lmalate concentrations, and with more citrate concentrations, in order to more definitively
conclude citrates mechanism of inhibition of fumarase.
Previous studies have found KI= 3.5 x 10-3.1 We found slight variation in the KI of fumarase
and citrate. KI (6.25 mM Citrate)= 5.79 x 10-3 and KI (18.5 mM Citrate)= 8.11 x 10-3.

Acknowledgements
We would like to thank Grinnell College and the Biological Chemistry Department for providing us
with the resources that made this project possible. Additionally, we would like to thank Professor
DeRidder and our TA, Tina Ding, for their support throughout the entirety of our experiment. We would
also like to thank members of our lab section for sharing kinetic data, as well as Bradford results.

References
1. DeRidder, B., Levandoski, M., Ortiz, C. Introduction to Biological Chemistry Laboratory Manual Fall
2016. Grinnell College.
2. Massey. Studies on Fumarase. 4. The Effects of Inhibitors on Fumarase Activity. Biochemical Journal
55.1 (1953): 172-177. Print.
3. Teipel and Hill. The Number of Substrate and Inhibitor Sites of Fumarase. Biochemical Journal 243. 10
(1968): 5679-5683. Print.
4. Weaver and Banaszak. Crystallographic Studies of the Catalytic and a Second Site in Fumarase C from
Escherichia coli. Biochemistry 35. 44 (1996): 13955-13965. Print.