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