526
14 Glycolysis
Activation by Fru-2,6-P2 may explain some
effects that previously had been attributed to
stable modifications of the enzyme. The PFK
from mammalian liver and muscle can in fact be
phosphorylated by a cAMP-dependent phosphokinase, but the catalytic or regulatory properties
of the enzyme are not significantly changed. Of
greater regulatory importance is the phosphorylation of the PFK from mammalian fat tissues, and
especially of the PFKs from the molluscs, nematodes and trematodes [14, 147]. The PFK of the
roundworm Ascaris suum is very similar to the
mammalian enzyme in its regulatory characteristics, despite the somewhat larger size of the subunits. However, the phosphorylation of a particular serine residue leads to a three- to fourfold
increase in activity under physiological conditions; this mainly involves a reduction in sensitivity to inhibitory ATP. The phosphorylation occurs
in vitro with a cAMP-dependent protein kinase
from bovine heart; Ascaris itself has two quite different protein kinases that are active on PFK, neither of which is influenced by cAMP or cGMP
[125]. Similar results have been obtained with the
PFK of the nematode Dirofilaria immitis [232].
The PFK of the liver-fluke Fasciola hepatica can
also be phosphorylated by the cAMP-dependent
mammalian enzyme and, as a consequence, has a
higher affinity for Fru-6-P and reduced inhibition
by ATP [147].
Fru-2,6-P2 is produced from Fru-6-P by a specific 6-phosphofructo-2-kinase, designated PFK-2
to distinguish it from the classical PFK (PFK-1).
The Fru-2,6-P2-forming enzyme of the mammals
cannot be separated from the Fru-2,6-Pz-cleaving
bisphosphatase (FBPase-2), and in fact sequence
data show that the 6-phosphofructo-2-kinase and
the fructose-2,6-bisphosphatase exist as domains
of the same polypeptide chain of 470 amino
acids; two such subunits make up a homodimer of
110 kDa. The kinase domains resemble PFK-1 in
their spatial structure, and amino acids 250-349
of the bisphosphatase are significantly homologous to phosphoglycerate mutase; thus, the
bifunctional enzyme apparently arose by gene
fusion [11, 139]. Fru-2,6-P2-synthesizing and
-cleaving enzyme activities are also found in the
same bifunctional enzyme protein in spinach leaves and in the flagellate Euglena gracilis; yeast,
however, has two separate proteins [54, 122].
Mammalian liver, muscle, heart and brain have
tissue-specific isoenzymes of the PFK-2IFBPase-2
with different functional and structural properties
[265]. The mammalian PFK-2, like the PFK-1, is
activated by AMP and inhibited by citrate and
phosphoenolpyruvate but, in contrast to PFK-1, it
is not inhibited by ATP. The phosphorylation of a
particular serine residue of the PFK-2IFBPase-2
of mammalian liver by a cAMP-dependent protein kinase leads to inhibition of the kinase and
activation of the bisphosphatase [121, 130]; the
phosphorylation is triggered, for example, by
glucagon or adrenalin. Phosphorylation of the
mammalian heart enzyme has exactly the opposite effect [121].
The complicated regulatoIY properties of the
animal PFKs evolved quite gradually. The enzymes of bacteria and the slime mould Dictyostelium show Michaelis-Menten kinetics with the
substrate Fru-6-P and no allost~ric effects. Sigmoidal Fru-6-P kinetics already exist for the PFKs
of certain protozoans, e.g. Leishmania histolytica, Crithidia fasciculata and Entamoeba histolytica, but, with the exception of AMP, none of the
usual modulators are effective. The PFK of Trypanosoma brucei shows little improved regulation
and, like other glycolytic enzymes, is bound to
the glycosome. This enzyme is tetrameric, like
other PFKs, but is the "odd-man-out" with small
subunits of only 49 kDa. AMP and ADP are stimulatory only at low concentrations, and are inhibitory at high concentrations. Citrate, Fru-1,6-P2,
Fru-2,6-P2 and Pi have no effect [43]. The PFK of
T. cruzi has similar characteristics [25]. The tissues
of some metazoans also contain PFKs on which
the typical effectors of the mammalian enzyme,
such as ADP, Fru-1,6-P2 and citrate, have no
effect; this is true, for example, in the roundworm Ascaris suum, the shell-adductor muscle of
the oyster Crassostrea virginica, and the flight
muscle of the cockroach Periplaneta americana
[244].
PFK as the key enzyme of glycolysis is adapted
to the specific internal and external conditions of
individual cells by species- and tissue-specific differences in regulatory properties. This becomes
apparent when one compares the heart muscle of
the rainbow trout Salmo gairdneri and the turtle
Pseudemys scripta. In the turtle heart, as in mammalian heart, hypoxia results in an increase in the
rate of glycolysis, and thus provision of ATP (Pasteur effect). This effect involves changes in the
concentration of allosteric modulators of PFK,
whereby the positive effectors AMP, ADP and Pi
become increased and the negative effectors ATP
and citrate are decreased. In trout heart, on the
other hand, the modulator sensitivity of PFK,
and thus the extent of the Pasteur effect, is much
lower; this organ is to a great extent adapted to
aerobic conditions [113]. A further example of the
14 Glycolysis
Activation by Fru-2,6-P2 may explain some
effects that previously had been attributed to
stable modifications of the enzyme. The PFK
from mammalian liver and muscle can in fact be
phosphorylated by a cAMP-dependent phosphokinase, but the catalytic or regulatory properties
of the enzyme are not significantly changed. Of
greater regulatory importance is the phosphorylation of the PFK from mammalian fat tissues, and
especially of the PFKs from the molluscs, nematodes and trematodes [14, 147]. The PFK of the
roundworm Ascaris suum is very similar to the
mammalian enzyme in its regulatory characteristics, despite the somewhat larger size of the subunits. However, the phosphorylation of a particular serine residue leads to a three- to fourfold
increase in activity under physiological conditions; this mainly involves a reduction in sensitivity to inhibitory ATP. The phosphorylation occurs
in vitro with a cAMP-dependent protein kinase
from bovine heart; Ascaris itself has two quite different protein kinases that are active on PFK, neither of which is influenced by cAMP or cGMP
[125]. Similar results have been obtained with the
PFK of the nematode Dirofilaria immitis [232].
The PFK of the liver-fluke Fasciola hepatica can
also be phosphorylated by the cAMP-dependent
mammalian enzyme and, as a consequence, has a
higher affinity for Fru-6-P and reduced inhibition
by ATP [147].
Fru-2,6-P2 is produced from Fru-6-P by a specific 6-phosphofructo-2-kinase, designated PFK-2
to distinguish it from the classical PFK (PFK-1).
The Fru-2,6-P2-forming enzyme of the mammals
cannot be separated from the Fru-2,6-Pz-cleaving
bisphosphatase (FBPase-2), and in fact sequence
data show that the 6-phosphofructo-2-kinase and
the fructose-2,6-bisphosphatase exist as domains
of the same polypeptide chain of 470 amino
acids; two such subunits make up a homodimer of
110 kDa. The kinase domains resemble PFK-1 in
their spatial structure, and amino acids 250-349
of the bisphosphatase are significantly homologous to phosphoglycerate mutase; thus, the
bifunctional enzyme apparently arose by gene
fusion [11, 139]. Fru-2,6-P2-synthesizing and
-cleaving enzyme activities are also found in the
same bifunctional enzyme protein in spinach leaves and in the flagellate Euglena gracilis; yeast,
however, has two separate proteins [54, 122].
Mammalian liver, muscle, heart and brain have
tissue-specific isoenzymes of the PFK-2IFBPase-2
with different functional and structural properties
[265]. The mammalian PFK-2, like the PFK-1, is
activated by AMP and inhibited by citrate and
phosphoenolpyruvate but, in contrast to PFK-1, it
is not inhibited by ATP. The phosphorylation of a
particular serine residue of the PFK-2IFBPase-2
of mammalian liver by a cAMP-dependent protein kinase leads to inhibition of the kinase and
activation of the bisphosphatase [121, 130]; the
phosphorylation is triggered, for example, by
glucagon or adrenalin. Phosphorylation of the
mammalian heart enzyme has exactly the opposite effect [121].
The complicated regulatoIY properties of the
animal PFKs evolved quite gradually. The enzymes of bacteria and the slime mould Dictyostelium show Michaelis-Menten kinetics with the
substrate Fru-6-P and no allost~ric effects. Sigmoidal Fru-6-P kinetics already exist for the PFKs
of certain protozoans, e.g. Leishmania histolytica, Crithidia fasciculata and Entamoeba histolytica, but, with the exception of AMP, none of the
usual modulators are effective. The PFK of Trypanosoma brucei shows little improved regulation
and, like other glycolytic enzymes, is bound to
the glycosome. This enzyme is tetrameric, like
other PFKs, but is the "odd-man-out" with small
subunits of only 49 kDa. AMP and ADP are stimulatory only at low concentrations, and are inhibitory at high concentrations. Citrate, Fru-1,6-P2,
Fru-2,6-P2 and Pi have no effect [43]. The PFK of
T. cruzi has similar characteristics [25]. The tissues
of some metazoans also contain PFKs on which
the typical effectors of the mammalian enzyme,
such as ADP, Fru-1,6-P2 and citrate, have no
effect; this is true, for example, in the roundworm Ascaris suum, the shell-adductor muscle of
the oyster Crassostrea virginica, and the flight
muscle of the cockroach Periplaneta americana
[244].
PFK as the key enzyme of glycolysis is adapted
to the specific internal and external conditions of
individual cells by species- and tissue-specific differences in regulatory properties. This becomes
apparent when one compares the heart muscle of
the rainbow trout Salmo gairdneri and the turtle
Pseudemys scripta. In the turtle heart, as in mammalian heart, hypoxia results in an increase in the
rate of glycolysis, and thus provision of ATP (Pasteur effect). This effect involves changes in the
concentration of allosteric modulators of PFK,
whereby the positive effectors AMP, ADP and Pi
become increased and the negative effectors ATP
and citrate are decreased. In trout heart, on the
other hand, the modulator sensitivity of PFK,
and thus the extent of the Pasteur effect, is much
lower; this organ is to a great extent adapted to
aerobic conditions [113]. A further example of the