trans

THE EMBDEN–MEYERHOF–PARNAS (EMP) PATHWAY OF GLYCOLYSIS 141
eukaryotic cells. Whereas in most cells the
glycolytic pathway takes place in the cytosol,
in the trypanosome its first seven enzymes are
localized within a membrane-bound organelle.
In addition, two enzymes of glycerol metabolism
and several enzymes of the hexosemonophosphate
pathway are present in these
organelles as well. Although this organelle
resembles the peroxisomes found in other cell
types, it lacks typical peroxisomal enzymes such
as peroxidase, catalase and acylCoA oxidase. By
contrast 90% of its protein represents glycolytic
enzyme. Therefore, these highly specialized
peroxisomes have been called ‘glycosomes’.
Because neither a functional tricarboxylic acid
cycle, nor a classical respiratory chain, are
present in the organism’s single mitochondrion,
the end-product of this metabolism is
pyruvate, which is excreted directly into the
host’s bloodstream rather than being metabolized
to lactate or carbon dioxide plus water.
Hence, little ATP is produced and that only
by substrate-level phosphorylation. This low
ATP yield explains the high rate of glucose
consumption (Figure 7.4). Bloodstream forms
contain neither carbohydrate stores, such as
glycogen or other polysaccharides, nor any
energy reserves of any significance, such as
creatine phosphate or polyphosphates. Thus
inhibition of the glycolytic pathway or depletion
of an energy substrate such as glucose
immediately results in a rapid drop of cellular
ATP levels, a total loss of motility and disappearance
of trypanosomes from the bloodstream
of the infected host.
The NADH produced during glycolysis
in the glyceraldehyde-phosphate dehydrogenase
reaction is reoxidized indirectly by
molecular oxygen via a glycerol-3-phosphate
(G3P):dihydroxyacetone-phosphate (DHAP)
shuttle comprising the glycosomal NAD-linked
G3P dehydrogenase and a FAD-linked G3P
dehydrogenase present in the mitochondrial
inner membrane, which catalyses the reversed
reaction. Reducing equivalents produced by
this cycle are supposed to react with ubiquinone
present in the mitochondrial membrane.
The ubiquinol thus formed is oxidized by a
ubiquinol:oxygen oxidoreductase, also called
trypanosome alternative oxidase (TAO), present
in the mitochondrial inner membrane. This
enzyme resembles the alternative oxidases of
plants and certain fungi in that it is insensitive
to cyanide, but is sensitive to salicyl hydroxamic
acid (SHAM). Moreover, in a phylogenetic
analysis its amino-acid sequence branched
close to the alga C. reinhardtii and formed a
single clade with the plant alternative oxidases
with a high bootstrap support. This enzyme has
also been detected in the plant trypanosomatid
Phytomonas, but seems to be absent from
Leishmania spp., where neither the activity nor
the gene encoding the enzyme were detected.
Inhibition of the TAO by SHAM mimics the
effect of a lack of oxygen on carbohydrate
metabolism of the bloodstream form. NAD is
now regenerated by the reduction of DHAP to
G3P, followed by the formation of glycerol which
is excreted by the organism. The ATP synthesized
in the latter reaction compensates for the
loss of one mole of ATP in the glycosomal phosphoglycerate
kinase reaction, because now one
mole of phosphoglycerate, rather than two, is
produced per mole of glucose consumed. As
a consequence, glucose is dismutated into equimolar
amounts of pyruvate and glycerol, with
net synthesis of one molecule of ATP. Under
these conditions bloodstream forms survive
and remain motile, while cellular ATP levels
drop to about 50%. Thus, inhibition of respiration
alone is not sufficient to kill the organism
and the trypanosomes survive for a prolonged
time as long as glycerol does not accumulate
in the medium. This probably explains why of
the many new and effective inhibitors of the
trypanosome alternative oxidase which have
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA