14.4 Phosphofructokinases
The phosphofructokinases (PFKs) transfer a
phosphate from ATP to fructose-6-phosphate.
With some PFKs, e.g. from mammals, the tapeworm Moniezia expansa and the flagellate Trypanosoma cruzi, ITP and GTP also function as
phosphate donors but, unlike ATP, are not effective as allosteric inhibitors [25]. The PFKs are
mostly present as tetramers with subunits of
35 kDa in the bacteria, 49 kDa in Trypanosoma
brucei, and between 80 and 95 kDa in the metazoans [43, 49]. The yeast enzyme is an octamer
with 100-kDa subunits; in the mammals, there is
aggregation to octamers and higher polymers, but
also dissociation to dimers [90]. The PFK of the
carrot has a size of 5 MDa but its 60-kDa subunits
are smaller than those of the yeast and mammals
[277]. Membrane-bound PFKs are known from
various mammals and the ciliate Tetrahymena;
these differ in their kinetic and regulatory properties from the soluble form [209]. The subunits of
PFK from rabbit muscle and mouse liver both
consist of 780 amino acids and agree 68 % in their
sequences [75]. The N- and C-terminal halves
show significant homology to each other and to
the 35-kDa bacterial enzyme. This supports the
concept that the mammalian PFK arose by
duplication and fusion of an ancestral PFK of
about half the size [196]. However, subdivision of
the rabbit PFK gene into 22 exons of 15-63
codons reveals no internal symmetry [135].
Tissue-specific PFK isoenzymes have been
detected in all vertebrates examined. Four types
of PFK subunit are known from humans and various other mammals, and these have different
chemical, immunological and regulatory characteristics. Heterotetrameric forms may arise where
different types are present in the same cell.
Muscle contains only subunit A (or M), which is
also predominant in other glycolytically active tissues such as the heart. Subunit B (or L) predominates in gluconeogenic organs such as the liver or
kidney cortex. The third type, known as C or For
P, is found in the brain, kidney, placenta, fibroblasts, erythrocytes and blood platelets. Embryonal cells initially express all three genes. The
tissue-specific pattern that arises later differs
between species or even between breeding lines.
Thus, human and rabbit brain contain all three
subunits, with A predominating in humans and C
predominating in the rabbit. Rat brain contains
mainly, or exclusively, type A, B or C, depending
upon the population examined [49,208]. It was
14.4 Phosphofructokinases
525
first discovered in 1983 that the gut mucosa has a
type-D enzyme with unique regulatory properties
[119].
Most comparative studies of PFK have concentrated mainly on the regulatory characters. The
results of in vitro experiments on mammalian
PFKs have given rise to a long list of low molecular weight substances that can function as activators, inhibitors or de-inhibitors. The binding of
calmodulin to rabbit muscle PFK confers calcium
sensitivity. In fact, PFK be looked upon as the
prototype of a multiply modulated enzyme. Mammalian PFKs display a series of common features
which are apparently valid for all vertebrates and,
in principle, also for many invertebrates [34, 125,
217,243, 244]. Positive cooperativity results in a
sigmoidal curve describing the dependence of
enzyme activity on the fructose-6-phosphate concentration; high ATP concentrations bring about
an allosteric inhibition. Citrate, phosphocreatine
or phosphoarginine, phosphoenolpyruvate and 6phosphogluconate, for example, are inhibitory.
AMP, cAMP, ADP, Pi> Fru-1,6-P2 and NH/ act as
positive modulators, reduce the cooperativity with
fructose-6-phosphate, and counteract the inhibition by ATP and citrate [100].
The activation of mammalian PFK by Fru-2,6P2 was first discovered in 1980 [107], and has since
been demonstrated in other PFKs, e.g. those in
amphibians, insects, molluscs, cestodes, nematodes, higher plants and fungi [34, 100, 188, 271,
272]. In contrast, the PFK of Trypanosoma cruzi
is not affected by Fru-2,6-P2 and that of the carrot
is affected only to a minor degree [254, 277]. Fru2,6-P2 is the most effective known activator, being
1000-fold more active than Fru-1,6-P2 with the
PFK of mammalian liver. At high concentrations
Fru-1,6-P2 prevents the stimulation by Fru-2,6-P2,
and in this case has an inhibitory effect. Fru-2,6P2 has not yet been clearly shown to have a significant regulatory function in mammalian
muscle; however, the Fru-2,6-P2 concentration
and glycolytic flux are closely correlated in
muscle of the frog Rana temporaria [273]. The different activators can have synergistic effects. The
four activators of the PFK present in the flight
muscles of the cockroach Periplaneta americana
at the physiological concentrations of 0.4 mmolll
AMP, 1 f-lmolll Fru-2,6-Pz, 3 mmolll NH4 +, and
10 mmolll Pi at pH 7 enhance the substrate affinity by 640-fold and reduce the affinity for the
inhibitor ATP by lO-fold. In this way, the halfmaximal PFK activity is achieved at 0.025 mmoIl
1, the in vivo concentration in muscle, instead of
at 16 mmolll [244].
The phosphofructokinases (PFKs) transfer a
phosphate from ATP to fructose-6-phosphate.
With some PFKs, e.g. from mammals, the tapeworm Moniezia expansa and the flagellate Trypanosoma cruzi, ITP and GTP also function as
phosphate donors but, unlike ATP, are not effective as allosteric inhibitors [25]. The PFKs are
mostly present as tetramers with subunits of
35 kDa in the bacteria, 49 kDa in Trypanosoma
brucei, and between 80 and 95 kDa in the metazoans [43, 49]. The yeast enzyme is an octamer
with 100-kDa subunits; in the mammals, there is
aggregation to octamers and higher polymers, but
also dissociation to dimers [90]. The PFK of the
carrot has a size of 5 MDa but its 60-kDa subunits
are smaller than those of the yeast and mammals
[277]. Membrane-bound PFKs are known from
various mammals and the ciliate Tetrahymena;
these differ in their kinetic and regulatory properties from the soluble form [209]. The subunits of
PFK from rabbit muscle and mouse liver both
consist of 780 amino acids and agree 68 % in their
sequences [75]. The N- and C-terminal halves
show significant homology to each other and to
the 35-kDa bacterial enzyme. This supports the
concept that the mammalian PFK arose by
duplication and fusion of an ancestral PFK of
about half the size [196]. However, subdivision of
the rabbit PFK gene into 22 exons of 15-63
codons reveals no internal symmetry [135].
Tissue-specific PFK isoenzymes have been
detected in all vertebrates examined. Four types
of PFK subunit are known from humans and various other mammals, and these have different
chemical, immunological and regulatory characteristics. Heterotetrameric forms may arise where
different types are present in the same cell.
Muscle contains only subunit A (or M), which is
also predominant in other glycolytically active tissues such as the heart. Subunit B (or L) predominates in gluconeogenic organs such as the liver or
kidney cortex. The third type, known as C or For
P, is found in the brain, kidney, placenta, fibroblasts, erythrocytes and blood platelets. Embryonal cells initially express all three genes. The
tissue-specific pattern that arises later differs
between species or even between breeding lines.
Thus, human and rabbit brain contain all three
subunits, with A predominating in humans and C
predominating in the rabbit. Rat brain contains
mainly, or exclusively, type A, B or C, depending
upon the population examined [49,208]. It was
14.4 Phosphofructokinases
525
first discovered in 1983 that the gut mucosa has a
type-D enzyme with unique regulatory properties
[119].
Most comparative studies of PFK have concentrated mainly on the regulatory characters. The
results of in vitro experiments on mammalian
PFKs have given rise to a long list of low molecular weight substances that can function as activators, inhibitors or de-inhibitors. The binding of
calmodulin to rabbit muscle PFK confers calcium
sensitivity. In fact, PFK be looked upon as the
prototype of a multiply modulated enzyme. Mammalian PFKs display a series of common features
which are apparently valid for all vertebrates and,
in principle, also for many invertebrates [34, 125,
217,243, 244]. Positive cooperativity results in a
sigmoidal curve describing the dependence of
enzyme activity on the fructose-6-phosphate concentration; high ATP concentrations bring about
an allosteric inhibition. Citrate, phosphocreatine
or phosphoarginine, phosphoenolpyruvate and 6phosphogluconate, for example, are inhibitory.
AMP, cAMP, ADP, Pi> Fru-1,6-P2 and NH/ act as
positive modulators, reduce the cooperativity with
fructose-6-phosphate, and counteract the inhibition by ATP and citrate [100].
The activation of mammalian PFK by Fru-2,6P2 was first discovered in 1980 [107], and has since
been demonstrated in other PFKs, e.g. those in
amphibians, insects, molluscs, cestodes, nematodes, higher plants and fungi [34, 100, 188, 271,
272]. In contrast, the PFK of Trypanosoma cruzi
is not affected by Fru-2,6-P2 and that of the carrot
is affected only to a minor degree [254, 277]. Fru2,6-P2 is the most effective known activator, being
1000-fold more active than Fru-1,6-P2 with the
PFK of mammalian liver. At high concentrations
Fru-1,6-P2 prevents the stimulation by Fru-2,6-P2,
and in this case has an inhibitory effect. Fru-2,6P2 has not yet been clearly shown to have a significant regulatory function in mammalian
muscle; however, the Fru-2,6-P2 concentration
and glycolytic flux are closely correlated in
muscle of the frog Rana temporaria [273]. The different activators can have synergistic effects. The
four activators of the PFK present in the flight
muscles of the cockroach Periplaneta americana
at the physiological concentrations of 0.4 mmolll
AMP, 1 f-lmolll Fru-2,6-Pz, 3 mmolll NH4 +, and
10 mmolll Pi at pH 7 enhance the substrate affinity by 640-fold and reduce the affinity for the
inhibitor ATP by lO-fold. In this way, the halfmaximal PFK activity is achieved at 0.025 mmoIl
1, the in vivo concentration in muscle, instead of
at 16 mmolll [244].