trans

148 ENERGY METABOLISM – TRYPANOSOMATIDAE
this enzyme seems to be absent from
Leishmania. T. brucei PGKs have an almost
absolute requirement for ATP, contrary to the
enzymes from yeast and rabbit muscle, which
accept GTP and ITP as well. The two major
trypanosome isoenzymes are inhibited by suramin,
but the glycosomal enzyme, with its
higher positive charge, is much more sensitive
to this inhibitor. The glycosomal isoenzyme has
been crystallized after removal, by genetic engineering,
of the C-terminal extension with PTS1,
responsible for its import into the glycosome,
and its structure solved. Comparison of the protein
sequences of the trypanosomatid enzymes
with those of other organisms has revealed
a typical prokaryotic origin of the former. The
enzyme must have entered the trypanosomatid
ancestor by horizontal transfer, but the donor
organism cannot, at present, be identified.
Phosphoglycerate mutase (PGAM)
and enolase
These two enzymes of T. brucei are present in
the cytosol and therefore they have received less
attention. The genes coding for both these proteins
now have been cloned and sequenced and
the corresponding proteins have been overexpressed.
Two classes of non-homologous PGAM
exist. All mammalian PGAMs use as cofactor
2,3-bisphosphoglycerate, while many bacterial
and plant PGAMs are cofactor-independent.
The T. brucei PGAM gene has been cloned and
sequenced and the enzyme expressed in E. coli.
The trypanosome enzyme turns out to be a typical
cofactor-independent protein and thus is
totally different from the isofunctional enzyme
of the host. It is closely related to the cofactorindependent
PGAMs found in the cytosol of
plants. Modeling of its three-dimensional structure
suggests that this class of proteins is related
to the family of alkaline phosphatases.
Pyruvate kinase (PYK)
The last enzyme of the glycolytic pathway is
pyruvate kinase. Like PGAM and enolase, PYK is
a cytosolic enzyme. The PYKs from T. brucei and
L. mexicana have been cloned, sequenced, overexpressed
in E. coli and purified. The enzyme
is activated by various sugar phosphates, most
notably fructose 2,6-bisphosphate, but also
by fructose 1,6-bisphosphate, glucose 1,6-
bisphosphate and ribulose 1,5-bisphosphate.
Its activity is modulated by adenine nucleotides
and inorganic phosphate, while it is insensitive
to regulation by most of the metabolites that
modulate PYK activity from other sources. The
structure of the L. mexicana enzyme has been
solved and reveals a pocket that contains a sulfate
molecule. It is this pocket that is thought
to be responsible for the binding of its allosteric
regulator fructose 2,6-bisphosphate. Based on
the comparison of the three-dimensional structure
of Saccharomyces cerevisiae PYK crystallized
with fructose 1,6-bisphosphate present at
the effector site (R-state) and the L. mexicana
enzyme crystallized in the T-state, two residues
(Lys453 and His480) have been proposed to
bind the 2-phospho group of the effector. These
predictions have subsequently been confirmed
by site-directed mutagenesis.
Pyruvate phosphate dikinase (PPDK)
In addition to an ATP-dependent PYK, T. brucei
also has a PP i -dependent pyruvate, phosphate
dikinase, or PPDK. The enzyme is encoded by
a single-copy gene. The gene product, a 100-
kDa protein, is expressed in procyclic forms
but not in the bloodstream forms of T. brucei.
The AKL motif at the C-terminal extremity of
PPDK has been shown to be essential for
glycosomal targeting. This contrasts with the
cytosolic PYK which is mainly active in the
bloodstream form. PPDK has been detected in
BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA