trans

THE EMBDEN–MEYERHOF–PARNAS (EMP) PATHWAY OF GLYCOLYSIS 145
with the human enzyme, which increases the
chances of developing specific inhibitors. The
latter enzyme is also twice as large owing to a
gene duplication that must have taken place
after the separation of the trypanosomatid lineage
from the main line of eukaryotic evolution.
Inspection of the sequence also suggests
that HK is routed to the glycosome by an
N-terminally located peptide that is reminiscent
of a peroxisomal import signal, also called
PTS2. The enzyme, which has a low specificity
towards sugars, is neither regulated by glucose
6-phosphate nor by glucose 1,6-bisphosphate,
as are the HKs from most other sources. Interestingly,
the enzyme has a low specificity for
ATP, and is also able to utilize other nucleotide
triphosphates such as UTP and CTP. This
absence of specificity probably reflects the
absence of such nucleotides from the matrix
of the glycosome and thus may render the
nucleotide-binding pocket of this enzyme an
interesting target for drug development. The
glucose-binding pocket offers an interesting
target as well. A number of glucosamine
analog inhibitors have been synthesized. The
most potent inhibitor, m-bromophenyl glucosamide,
did not affect the activity of HK
from yeast but inhibited the growth of live trypanosomes
in vitro. The mode of action of the
m-bromophenyl glucosamide inhibitor on the
trypanosome HK has been elucidated by modeling
the structure of the T. brucei enzyme.
Glucose-6-phosphate isomerase (PGI)
In the bloodstream form PGI is mainly associated
with the glycosome. It catalyses an equilibrium
reaction and is often considered not
to confer any significant control over the glycolytic
flux. However, any efficient PGI inhibitor
would be able to cause arrest of growth. In this
respect the potent transition-state analog
5-phospho-D-arabinonate is an interesting
candidate. This compound binds to the enzyme
with a K i of 50 nM, while a K i /K m value of 2000
was obtained. Such a tight binding inhibitor
may compete effectively with the high concentration
of intermediates inside the glycosome.
Its mode of binding to the active site of the
homologous rabbit enzyme has been solved at
1.9 Å resolution, and the compound mimics
the cis-enediol intermediate of the catalytic
reaction. Unfortunately, due to the nature of
such an inhibitor, there was little specificity
for the trypanosome enzyme when compared
to the yeast PGI.
Phosphofructokinase (PFK)
T. brucei PFK, as well as the Leishmania donovani
enzyme, has been cloned, sequenced
and overexpressed. The deduced polypeptide
sequences both contain a peroxisome targeting
signal present at the C-terminus (so-called
PTS1). Although their respective sequences
predict a typical inorganic pyrophosphate
(PP i )-dependent PFK, both have an absolute
requirement for ATP and are completely inactive
with PP i as substrate. Apparently, during
evolution this trypanosomatid enzyme must
have changed its specificity. Another difference
from the PFKs from other eukaryotes is
that it is not regulated by fructose 2,6-bisphosphate.
The two trypanosomatid enzymes are
inhibited by AMP and furthermore have similar
properties. Contrary to the situation in mammalian
cells, the T. brucei PFK exerts hardly
any control over the glycolytic flux. While from
a rate-controlling point of view PFK would be
less interesting for drug design, its remarkable
structural difference from the host enzyme
renders it a very interesting candidate indeed.
Aldolase
This enzyme belongs to the class I aldolases that
catalyse the reversible cleavage of the fructose
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA