trans

146 ENERGY METABOLISM – TRYPANOSOMATIDAE
1,6-bisphosphate, via the formation of a Schiff
base, into the trioses dihydroxyacetone phosphate
(DHAP) and glyceraldehyde 3-phosphate
(GA3P) without the need of a divalent metal ion.
The enzyme is abundantly present in T. brucei
and exerts a significant amount of control over
the glycolytic flux, which does not seem to be
the case for the red blood cell, where deficiencies
seem to have little or no effect. The aldolase
gene of both T. brucei and L. mexicana has
been cloned and sequenced and the predicted
polypeptide sequence contains an N-terminal
PTS2. In a recent phylogenetic analysis the two
trypanosomatid sequences formed a monophyletic
group with the aldolases found in the
chloroplasts from both algae and plants, where
the trypanosomatid occupied a sister position
relative to the green and red algae.
Triose-phosphate isomerase (TIM)
The trypanosome TIM was the first parasite
enzyme to be crystallized and its threedimensional
structure solved. This enzyme
catalyses the reversible conversion of the
triosephosphates DHAP and GA3P. In the
bloodstream form the enzyme is mainly found
in the glycosome, and it represents less than
0.04% of the total trypanosome protein. In general
few inhibitors of TIM have been described.
The trypanosome enzyme is inhibited by sulfate,
phosphate, arsenate, and by 2-phosphoglycolate.
Suramin also seems to bind to
T. brucei TIM, and there is some evidence that
suramin may interfere with the formation of
the active homodimeric form of the enzyme.
Although there is no theoretical argument to
suggest that inhibition of the enzyme would
lead to the death of the parasite, knockout
experiments have shown that TIM is a vital
enzyme and thus may be a good drug target.
TIM is the only glycolytic enzyme found
in glycosomes for which no (putative)
peroxisome-import signal has been reported
so far. Import studies into glycosomes have
shown that a 21 amino-acid-long internal peptide
has the ability to route a reporter protein
to glycosomes. Replacement of this peptide by
a corresponding peptide of the yeast TIM completely
abolished import. It is believed that this
peptide, which is exposed at the surface of the
native protein, mediates the interaction of TIM
with another protein that does carry a peroxisome
targeting signal (PTS). Import of TIM into
glycosomes would then be a piggyback process.
Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH)
All Trypanosomatidae studied, including T. brucei,
have two separate GAPDH isoenzymes, one
in the cytosol and one in the glycosome. While
the glycosomal enzyme is directly involved in
glycolysis, the function of the cytosolic isoenzyme
is not yet clear. The glycosomal isoenzyme
is inhibited by the epoxide-containing GAPDH
inhibitor pentalenolactone and by suramin,
gossypol and agaricic acid. Although its
substrate-binding site is well conserved, it has
been possible to synthesize epoxide or alphaenone
containing analogs that selectively and
irreversibly inhibit not only the enzyme but
also the growth of the trypanosome in an
in vitro culture system. The three-dimensional
structures of the T. brucei, T. cruzi and L. mexicana
enzymes have been solved, and this has
revealed a binding site for the NAD cofactor
which has some interesting differences from
the corresponding binding site in the human
enzyme, responsible for a reduced affinity for
this cofactor. A number of highly selective
adenosine analogs have been synthesized that
tightly fit the NAD-binding pocket of the
trypanosome enzyme, but do not bind to the
corresponding pocket of the mammalian
homolog. Some of these compounds have a
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA