Biochemical

Education
Pergamon Biochemical Education 26 (1998) 73-76

A simple experiment demonstrating the allosteric regulation of yeast

pyruvate kinase
Richard L. Taber, Angela Campbell, Scott Spencer
Department of Chemistry, The Colorado College, Colorado Springs, CO 80903, U.S.A.

Abstract

A simple experiment in which the regulatory properties of yeast pyruvate kinase are determined. Students do a partial purification
using PEG precipitation and find that fructose-l,6-bisphosphate activates the enzyme. ATP and alanine negatively modulate its
catalytic activity. The entire experiment can be done in one laboratory period using simple equipment. © 1998 IUBMB. Published
by Elsevier Science Ltd. All rights reserved.

I. Introduction while A T P and alanine negatively inhibit the rate. An

A simple experiment which illustrates the regulation
of an enzyme is a valuable part of an undergraduate
biochemistry laboratory program. To achieve this,
Hannappel developed an elegant experiment showing
the effects of metabolites on an enzyme reaction rate
using partially purified rat liver pyruvate kinase [1 ]. In his
experiment, the students were provided a rat liver extract
and they measured the effect of fructose 1,6-bisphos-
phate, alanine and A T P on the rate of the conversion of
phosphenol pyruvate (PEP) to pyruvate. Although the
students were capable of partially purifying the rat liver
pyruvate kinase themselves, due to the large numbers of
students involved this was done by technicians.
We found that our students had major objections to
sacrificing rats, whether they did it themselves, or if it was
done by a technician, simply to provide an enzyme for an
undergraduate experiment. So, we have substituted
pyruvate kinase obtained from Fleischmann's Active
Dried yeast for the rat liver pyruvate kinase, obtaining
results similar to those reported by Hannappel.
In our experiment, the students ground Fleischmann's
Active Dry yeast obtained from a local market in a
mortar and pestle and partially purified the pyruvate
kinase by a polyethylene glycol (PEG) precipitation of
the extract. The students then examined the effect of
fructose 1-6-bisphosphate, ATP and alanine on the rate
of conversion of PEP to pyruvate. They were able to
accomplish this work in a single laboratory session. The
addition of fructose 1-6-bisphosphate to the pyruvate
kinase assay medium results in a rate enhancement,
0307-4412/98/$19.00 © 1998 IUBMB. Published by Elsevier Science Ltd. All rights reserved.
PII: S0307-44 12(97)00 1 17-9
added educational feature of the experiment is that
studying the rate of the pyruvate kinase reaction requires
the use of a coupled assay, in which the product pyruvate
(PA) is reduced by N A D H to lactate using lactic
dehydrogenase. The rate of reaction is monitored by
measuring the disappearance of the N A D H absorption
at 340 nm.
PA kinasc
PEP+ADP > ATP+PA+NADH
LDH
• Lactate+NAD ÷ (1)
Yeast pyruvate kinase has a regulatory function in
coordinating glycolysis and gluconeogenesis [2-5]. The
pyruvate kinase reaction is irreversible in the PEP to
pyruvate direction. Figure 1 shows that when glycolysis is
operative producing fructose 1,6-bisphosphate (FBP),
pyruvate kinase needs to be active, so that the carbon is
sent to pyruvate. Yeast pyruvate kinase, like the
mammalian liver enzyme, shows little activity unless
fructose 1,6-bisphosphate is present in concentrations
sufficient to activate it. During gluconeogenesis, ATP
levels in the cell are high and oxaloacetate is converted to
PEP. The pyruvate kinase is turned off by negative
inhibition with ATP so that the carbon can be converted
back to glucose rather than diverted to pyruvate, which
would create a futile cycle. Often, alanine, after trans-
amination to pyruvate is a source of the carbon for
increasing the concentration of oxaloacetate for gluco-
neogenesis; thus, alanine also inhibits pyruvate kinase
preventing the newly synthesized PEP from going back
to pyruvate.
74 R. L. Taber et al./Biochemical Education 26 (1998) 73-76
GLUCOSE
1l (Two steps)
FRUCTOSE-6-PHOSPHATE
1L
PHOSPHOENOL PYRUVATE (PEP)
Pyruvate Kinase
Gluconeogenesls / PYRUVATE
OXALOACETATE ALANINE
Fig. 1. Pyruvate kinase regulates the irreversible conversion of PEP to
pyruvate. When phosphofructokinase is active producing fructose
1,6-bisphospbate, this molecule activates pyruvate kinase sending the
carbon to pyruvate. When the cell ATP levels are high, both phospho-
fructokinase and pyruvate kinase are inactivated. During gluconeo-
genesis, pyruvate kinase must be inactivated so that a futile cycle does
not result. Thus, both high alanine and ATP levels inactivate pyruvate
kinase. If glycolysis is stimulated, the fructose 1,6-bisphosphate can
override the negative inhibition by ATP or alanine.
2. Experimental
All reagents were purchased from Sigma Biochemi-
cals. Spectrophotometric determinations were made at
room temperature with a Milton Roy Spectronic 21
spectrophotometer. Protein quantitation was accom-
plished using the Bradford method [6].
Pyruvate kinase was isolated from Fleischmann's
Active Dry Yeast by grinding 10 g of yeast with a mortar
and pestle containing 20ml of 0.1M potassium
phosphate buffer, pH 7.6, which also contained MgCl2
(5 mM), EDTA (5 raM), PMSF (2mM), 2-mercapto-
ethanol (40 mM) and a teaspoon of washed sand. All
steps were performed at 0°C. The suspension was centri-
fuged at 20000g for 15min and the supernatant
Table 1
Yeast pyruvate kinase assay. Pipette the following reagents into a 3 ml spectrophotometric tube. Start the reaction by adding the enzyme solution. For
kinetic studies without the fructose 1,6-bisphosphate add 0.1 ml to the water
Reagent
Water
0.1 M phosphate buffer, pH 7.6 (buffer A)
8 mM phosphoenol pyruvate, monopotassium salt
3 mM NADH, disodium salt (in buffer A)
100 mM Mg(SO4)
40 mM ADP, di(monocyclohexylammonium salt)
L-lactic dehydrogenase (500 units/ml in buffer A)
30 mM fructose 1,6-bisphosphate, disodium salt
Enzyme solution
collected. The supernatant was brought to a 6% PEG
(w/w) concentration by stirring in dropwise a solution of
40% (w/w) PEG 8000 (Sigma #P-2139), centrifuged at
10000g for 10 min and the supernatant collected.
The pyruvate kinase activity was determined by
measuring at room temperature the NADH absorbance
decrease at 340 nm using the assay outlined in Table 1,
which is adapted from that published by Sigma [7].
Without fructose 1,6-bisphosphate (FBP) present, yeast
pyruvate kinase shows little activity. The ingredients of
the assay were placed in a spectrophotometric tube,
mixed thoroughly, the tube was placed in the spectro-
photometer and the first value read at 15 s. The absorb-
ance was recorded by taking readings every 15 s for 60 s,
usually with little change in absorbance being observed.
The sample was removed from the spectrophotometer
and 50 #1 of 30 mM fructose 1,6-bisphosphate was added
to activate the enzyme. The assay tube was mixed
thoroughly, placed back into the spectrophotometer and
the first absorbance recorded 15 s after the addition of
the FBP (75 s after the start of the reaction), with
readings taken for another 60-75 s. With the addition of
the FBP there should be a noticeable increase in the rate
of reaction as measured by a decrease in absorbance. In
our work, the enzyme extract from the PEG partial
purification was diluted 1:3 so that the absorbance
change after adding the FBP was about 0.2 units over a
60 s period. Other investigator's preparations could have
different pyruvate kinase activity. If there is not a suffi-
cient change in absorbance, then more enzyme extract
should be added, adjusting the amount of water to keep
the total volume in the assay tube at 3 ml.
If the change in absorbance after the addition of FBP
is judged to be about right (see Fig. 2), the effect of ATP
can be observed by removing the assay tube from the
spectrophotometer at about the 150s point, adding
100/A of 120 mM ATP and recording the change in
absorbance, as previously described, for about another
60 s. The effect of FBP on reversing the ATP inhibition
can be accomplished by removing the spectrometer assay
tube, adding 100/d of FBP solution and again recording
the absorbencies. In a second run, the inhibitory effect of
Volume in sample (ml) Volume in blank (ml)
1.30 1.40
0.80 0.80
0.16 0.16
0.20 0.20
0.20 0.20
0.10 0.10
0.04 0.04
0.10 0.10
0.10
 R. L. Taber et al./Biochemical Education 26 (1998) 73-76 75

08 Addition of ATP causes a marked decrease in the

enzyme activity. However, if the FBP is added again to
07
the ATP containing reaction mixture, the inhibition is
06
50 ul FBP
reversed. Fig. 3 shows that L-alanine inhibits the pyruvate
kinase, but it appears to be a weaker inhibitor than ATP.
0.5
Addition of FBP also reverses the L-alanine inhibition.
E ~ 100 ul ATP
~ 04
< • l O O ul F B P

03
~..~DO ul ATP 4. Discussion
02

01 The issue of whether laboratory animals should be

sacrificed for scientific studies is a major topic among our
0 , " " ' : ' " ' : " " " : ' " " : ' ' ' I ' ' ' I ' ' ' I

60 120 180 240 300 360 420

students. While most would agree that animal sacrifice is
Time (seconds) acceptable when significant advances for human health
Fig. 2. AIIosteric regulation of yeast pyruvate kinase. The reaction was are likely to result from the study, many would not agree
started by adding 100/d of a 1:3 dilution of the yeast extract from the to kill laboratory rats for educational experiments when
6% PEG precipitation. Addition of 50pl of 3 0 m m fructose non-mammalian alternatives are available. Mammalian
1,6-bisphosphate (FBP) activates the enzyme. Addition of 100/d of liver pyruvate kinase plays an important role in
120 mm A T P inhibits the reaction. The A T P inhibition was reversed by
addition of 100/2 of 30 mm FBP.
regulating the flow of the carbon in glycolysis and gluco-
neogenesis, and it is easy to obtain and for students to
study the effects of modulators on its activity. Fortu-
L-alanine was determined by injecting the FBP nately, yeast pyruvate kinase can be substituted for the
containing pyruvate kinase solution with 50mm rat liver enzyme when performing experiments for the
L-alanine and measuring the effect on the rate of training of students; the yeast can be purchased locally,
reaction as shown in Fig. 3. The stimulatory effects of the partial purification is very easy to accomplish and a
FBP and the inhibitory effects of ATP and L-alanine variety of kinetic experiments can be accomplished with
could be demonstrated all in a single run. simple equipment.
Our students were relatively experienced in under-
graduate enzyme kinetics when they performed this
3. Results experiment, so they were able to accomplish the pyruvate
kinase isolation, partial purification and kinetic study in
Figure 2 shows that yeast pyruvate kinase has little one four-hour laboratory period. For laboratory sessions
activity until it is activated by fructose 1,6-bisphosphate. which are shorter, the experiment can be modified. Our
partial purification increased the specific activity of the
pyruvate kinase 4-8-fold, providing more than enough
O8
activity for several students to do the kinetic study. The
07 laboratory instructor could perform this operation
before the laboratory session. The supernatant from the
0.6

50 ul FBP
centrifuged crude yeast extract has significant pyruvate
0.5
5O ul A l a
kinase activity, so the partial purification step might be
eliminated, although a greater quantity of the enzyme
~ 04 50 ul Ala
might have to be added to the assay mixture to give
< 100 ul Ala
1 0 0 u[ F B P
0.3 sufficient changes in absorbance in the kinetic studies.
02
The experiment could be accomplished in two labora-
tory periods. During the first period, the pyruvate kinase
01 could be isolated from the yeast and partially purified
0 , ' ' ' ~ ' ' ' ~ ' ' ' : " " " : " " " ; " " " : " " " : " " ~ I
with the purification being monitored by measuring the
60 120 180 240 300 360 420 480 specific activity and S D S - P A G E . When we stored the
Time (seconds)
supernatant from the 6% PEG precipitation at - 2 0 ° C
Fig. 3. Inhibition of yeast pyruvate kinase by L-alanine. The reaction for a week, about 30% of the original pyruvate kinase
was started by adding 100/d of a 1:3 dilution of the yeast extract from activity pyruvate remained, sufficient to perform the
the 6% PEG precipitation. Some preparations showed brief initial
activity without addition of the FBP activator. Successive additions of
kinetic experiments.
30 mm L-alanine slowed the reaction considerably. Addition of 100 id The experiment can be expanded; our students
of 30 mm fructose 1,6-bisphosphate reversed the L-alanine inhibition discovered that while adding FBP reversed the ATP
76 R. L. Taber et aL/Biochemical Education 26 (1998) 73-76
inhibition, adding ADP did not. Hannappel [1] suggested
that the stereospecificity of alanine binding be investi-
gated by substituting D-alanine for L-alanine. We have
had students show that pyruvate kinase gives a sigmoidal
rate curve with respect to PEP by measuring the rate of
reaction and plotting it against a concentration of PEP
over a range of 0.5-10 mM. When these measurements
are repeated in the presence of 1 mM FBP, the rate curve
v e r s u s PEP concentration no longer is sigmoidal. These
rate-curve experiments likely are for more advanced
students; many more solutions need to be prepared and
numerous kinetic measurements are required.
References
[1] E. Hannappel, W. Filcher, K. Brand, Biochem. Educ. 15 (1987)
42-43.
[2] C.N. Morris, S. Ainsworth, J. Kinderler, Biochem. J. 234 (1986)
64 ! -647.
[3] C.N. Morris, S. Ainsworth, J. Kinderler, Biochem. J. 234 (1986)
691-698.
[4] J. Kinderler, S. Ainsworth, C.N. Morris, N. Rhodes, Biochem. J.
234 (1986) 699-703.
[5] S. Ainsworth, Biochem. J. 234 (1986) 705-714.
[6] M.M. Bradford, Anal. Biochem. 72 (1976) 248-254.
[7] Sigma Biochemicals Enzyme Assay Protocols, www.sigma.sial.com/
sigma/techlib.htm.