Biochemistry and Molecular Biology Education 28 (2000) 248}250

A simple setup to illustrate metabolic pathway dynamics

Narayan S. Punekar*
Biotechnology Centre, Indian Institute of Technology * Bombay, Powai, Mumbai 400 076, India

Abstract

The sense of dynamic nature of a metabolic pathway, particularly in its steady state, is the most di$cult to convey to the student.
Such insights into metabolism have come from extensive and cumulative experimental data. These are not easily amenable to
laboratory demonstrations. A working model is described here to illustrate metabolic concepts in a laboratory and to complement
classroom teaching.  2000 IUBMB. Published by Elsevier Science Ltd. All rights reserved.

1. Introduction illustrate how metabolic pathways (and their control) are

The study of intermediary metabolism at one time was
the centerpiece of biochemistry and cellular physiology.
This approach brought into focus the pivotal role of
enzymes as instruments of cellular activities. The
present renewal of interest in the study of metabolism is
due to the potential of rational metabolic/pathway
engineering.
I have come across di!erent approaches to appreciate
metabolism as a student and as a teacher. Brie#y, they
include: (a) Simply write down and memorize all the
steps/events of a given metabolic pathway by rote.
Understandably such an encounter with metabolism
makes it a dull and intimidating subject. Often this ex-
perience turns out traumatic to the student. With the
advent of computers and easy access to well-organized
databases, there has been an added premium on memor-
izing. (b) Problem/ concept-based teaching of metabol-
ism. The approach brings out the metabolic logic in
chemical and functional terms. This allows us to appreci-
ate the intricate details of organization and major themes
of regulation in metabolism. Time constraints however
permit a selective emphasis on principle pathways and
representative modes of metabolic control. (c) Tech-
niques/methods-based approach. Here the pathway(s) in
question are under-emphasized over the concepts; stu-
dent is made to learn the pathways indirectly. One can
choose from a variety of examples in the literature to
* Tel.: #91-22-576-7775; fax: #91-22-572-3480.
E-mail address: nsp@btc.iitb.ernet.in (N.S. Punekar).
unraveled [1].
The sense of dynamic nature of a metabolic pathway,
particularly in its steady state, is the most di$cult to
convey to the student. Understandably, such insights
into metabolism have come from extensive and cumulat-
ive experimental data. This means there are fewer exam-
ples amenable to laboratory teaching (within the time
constraints) of metabolism. Analogies to provide a tour
de force of metabolism include- dams on a river [2,3],
#uid #ow through pipes/ tubes [3,4] and #ow of vehicu-
lar tra$c [5]. These metaphors cannot be adequately
translated into a working model to illustrate metabolic
concepts in a laboratory. This stimulus led me to develop
a setup in 1997 and to complement my classroom teach-
ing with a model demonstration.
2. The apparatus
The requirements to assemble the apparatus are; glass
beakers with inlet and outlet (modi"ed by simple glass
blowing), #exible tubing, connectors and stopcocks, mag-
netic stirrers and stir-bars, peristaltic pump (with adjust-
able #ow rate), wooden blocks, water soluble dye or a pH
indicator (e.g. bromocresol purple) and a reservoir for
water. A typical setup representing four metabolites
A through D, in a linear pathway, is shown in Fig. 1.
Water in each beaker represents a metabolite while the
tubes connecting beakers may be viewed as enzymatic
steps. The amount of water in each beaker corresponds
to a unique metabolic pool size. The pump could be
viewed as the driving force for the pathway from A to D.
In the time frame of the demonstration, the reservoir may

1470-8175/00/$20.00  2000 IUBMB. Published by Elsevier Science Ltd. All rights reserved.
PII: S 1 4 7 0 - 8 1 7 5 ( 0 0 ) 0 0 0 4 1 - 2
 N.S. Punekar / Biochemistry and Molecular Biology Education 28 (2000) 248}250
Fig. 1. Schematic representation of the apparatus for a four-metabolite case.
be viewed as an unlimited supplier of water. The stirrer at
C will ensure a proper mixing of the incoming dye solu-
tion from B.
3. An approach to concepts
Di!erent metabolites in a pathway are connected and
there is a #ow of matter. Showing that water from one
beaker ( held at a higher level) #ows into the other and so
on can simulate this. Inhibition of a step by backing up of
accumulated product is a very important phenomenon
but is often under-emphasized. Raising the level of water
in D can show this; a reversal of #ow from D to C is also
observed. A connector with a valve can mimic an irre-
versible step in the sequence. However a T-connector
from A and leading to two separate beakers represents
a branch point (not shown in Fig. 1).
For a metabolic reaction sequence to function, the
system normally has to overcome two kinds of barriers
* kinetic and thermodynamic. Enzymes are eminently
suited to help climb kinetic hills. Inputs of energy (free
energy!) are needed to surpass thermodynamic barriers.
The peristaltic pump and its position in the sequence can
illustrate this point. Flux through biosynthetic pathways
is often sustained by continuous product removal. The
pump located at the end of the sequence (as shown in
Fig. 1) represents this case. Most catabolic pathways
are driven by the availability of high concentrations of
the catabolic substrate (pump placed before A). Finally,
the #ux-generating step (a thermodynamically downhill
reaction) or a rate determining step could interrupt
a series of reactions that are at or near equilibrium. By
adapting the pump between them, BPC can be shown
as such a step in the sequence.
The concept of steady state can be demonstrated by
regulating the pump. When the in#ow equals the out#ow,
the water level in the four beakers is maintained (Fig. 1).
Keeping the levels same, the #ux through the sequence
can be increased by an equal and simultaneous increment
249
of input and output. The rate of net #ow of matter can
take di!erent values while the system continues to be at
steady state. A transient break in the #ow of water into
A will lead to a decrease in its level in all the beakers.
A steady state could now be reestablished even while the
water level is di!erent (a di!erent set of pool sizes for
A}D). Unique steady states can be thus achieved by
setting di!erent #ux rates and pool sizes.
The idea of di!erent pool sizes for metabolites can be
addressed by simply varying the elevation (using wooden
blocks) of each beaker. Maintaining the rate of #ow from
BPC at di!erent levels of B would illustrate a case for
the lack of correlation between metabolite concentration
and the reaction rate. As can be seen in Fig. 1, pool size of
C is the smallest while that of B is the largest. The
smallest limiting value for C will really mean B going
directly to D * a possible case to represent metabolic
channeling.
The setup as shown in Fig. 1 can be used to simulate
a metabolic crossover study. Blocking the #ow from B to
C (inhibition of BPC step) will lead to a decline in the
water levels at C and D without a!ecting the levels in
A and B; a continued operation of the pump will event-
ually empty the two beakers. The crossover point will be
obviously between B and C. At yet another level, students
could be asked to predict the e!ect of a known metabolic
inhibitor (such as #uoride). For example, the setup in
Fig. 1 can be rearranged to place the pump between
beakers A and B. The four beakers labeled as 1,3-bi-
sphosphoglycerate (A), 3-phosphoglycerate (B), 2-phos-
phoglycerate (C) and phosphoenolpyruvate (D) can now
represent a segment of glycolytic sequence. Fluoride in-
hibits the enolase reaction [2]. This is equivalent to block
in #ow from CPD. Such a block causes a metabolic
backup and accumulation of 2-phosphoglycerate (C) and
3-phosphoglycerate (B). However, since the pump is be-
tween A and B, the level of A is una!ected by `#uoridea
inhibition.
The idea of using a dye (and monitoring its absorb-
ance) to demonstrate precursor-product relationship was
250 N.S. Punekar / Biochemistry and Molecular Biology Education 28 (2000) 248}250
adopted from a book [6]. The dye mixed into B (Fig. 1
and with the pump on) will "rst be seen in C and only
later in D. Of course, the highest concentration of the dye
recorded in C will always be lower than that added to
B initially. Similarly, the highest concentration of dye in
D can never be greater than that of C [6]. Unless there is
a #ux reversal the dye will not "nd its way into A. An
interesting variant of this is possible with longer tubing
connecting the four beakers. These could be so arranged
that it is di$cult to make out which is connected to
which. A repeat of the above dye tracer exercise would
make it possible to identify beakers A}D (i.e. the ordering
of metabolites). The exercise is a suitable metaphor to
illustrate the `OrnithinePCitrullinePArgininosucci-
natePArgininea sequence of the arginine biosynthetic
pathway. It can be readily complemented with the
popular genetic data (of Beadle and Tatum fame) on
auxotrophic mutants.
The above examples could be conveyed as an e!ective
supplement to a theory course in metabolism and
regulation. Once assembled, the apparatus can be used
repeatedly without any signi"cant consumable compon-
ent. Although a few analogies mentioned here appear
relatively stretched * they have their merit in presenting
dynamic aspects of metabolism. With a bit of imagin-
ation, obviously many more metabolic situations can be
visualized through this model.
References
[1] S. Dagley, P.J. Chapman, Methods Microbiol. 6A (1971) 217}268.
[2] D. Voet, J.G. Voet, Biochemistry, Wiley, New York, 1990.
[3] E.A. Newsholme, C. Start, Regulation in Metabolism, Wiley Inter-
science, New York, 1981.
[4] G.D. Weston (Ed.), Biosynthesis and the Integration of Cell Meta-
bolism, BIOTOL Series, Butterworth & Heinmann, Oxford,
1992.
[5] D. Fell, Understanding the Control of Metabolism, Portland Press,
London, 1997.
[6] I.H. Segel, Biochemical Calculations, 2nd Edition, Wiley, New
York, 1976.