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THE EMBDEN–MEYERHOF–PARNAS (EMP) PATHWAY OF GLYCOLYSIS 143
been developed, and of which ascofuranone
was the most potent, none has been developed
into an effective anti-trypanosome drug.
Once glycerol accumulates above several
millimolar in the presence of a TOA inhibitor,
this metabolite becomes highly toxic to the
trypanosome. Due to mass action the reversal
of the glycerol-kinase reaction comes to a halt
and glycolysis stops. Since trypanosomes lack
energy stores, cellular ATP levels rapidly drop to
zero, leading to their complete immobilization.
Under these conditions trypanosomes are rapidly
cleared from the blood of infected animals.
Based on the above observations, there is
little doubt that the enzymes of glycolysis in
the bloodstream form of T. brucei constitute
excellent chemotherapeutic targets, but few of
these have been validated by gene knockout
experiments. Moreover, there is much discussion
as to which of the enzymes constitute the
better targets within the pathway. Mathematical
modeling experiments, using all the available
information on the individual enzymes
of this pathway, were able to predict successfully
the experimentally determined fluxes and
metabolite concentrations in trypanosomes.
This has allowed us to get information about
the degree of control each of the enzymes
exerts on the overall carbon flux through the
pathway. The glucose transporter protein that
is present in the parasite’s plasma membrane
may confer 50% or more of the control over
the pathway. However, the enzymes aldolase,
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), glycerol-3-phosphate dehydrogenase
(G3PDH) and phosphoglycerate kinase
(PGK) exert a considerable amount of control,
especially under conditions where the blood
levels of glucose are not limiting. Tight-binding
inhibitors of enzymes that confer control over
the pathway, with affinities in the nanomolar
range, as well as irreversible inhibitors of
enzymes that do not confer any control, may
be effective in killing the organism, provided
they succeed in inhibiting the flux to a large
extent. That this approach is feasible has
been illustrated by the use of conditional
knockouts of T. brucei cell lines. When the
activity of triosephosphate isomerase (TIM)
was decreased to 15%, the rate of cell division
was halved. Lower TIM levels were lethal. This
result was surprising since TIM was considered
a non-essential enzyme for trypanosome glycolysis.
Its inhibition (or removal) would force
the trypanosome into the formation of equimolar
amounts of pyruvate and glycerol with a
net production of only one ATP per molecule
of glucose consumed, rather than the two molecules
of ATP produced under normal conditions.
Yet the removal of TIM leads to arrest of
growth. Apparently the reduced amount of ATP
produced under these conditions may not be
sufficient to sustain cell division, but other
explanations, such as the accumulation of certain
toxic metabolites, cannot be excluded.
Similarly, in vitro growth in the presence of
low concentrations of SHAM, which imposes
an anaerobic type of glycolysis upon the organism,
turned out to be impossible over extended
periods as well. Upon lowering the level of TAO
expression by a conditional gene knockout, the
oxygen consumption was reduced fourfold and
the rate of trypanosome growth was halved. This
indicates that any glycolytic inhibitor that could
exert a considerable reduction of the overall glycolytic
flux, and/or a reduction of the ATP yield
of glycolysis, would arrest growth completely
and thus could be a promising drug candidate.
Bearing this in mind, each of the individual
steps of the glycolytic pathway will be discussed.
Metabolite transporters
Glycolysis proceeds at very high rates (up to
100 nM of glucose consumed per min and
per mg of protein) in the bloodstream form of
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA