530
14 Glycolysis
PEP metabolism must be introduced; this is especially the case in many invertebrates [173].
14.7.1 Pyruvate Kinase Isoenzymes
of Mammals
Mammals and birds have four tissue-specific
pyruvate kinase isoenzymes. All the activity in
skeletal muscle and most of the activity in heart
muscle and the brain stems from PK-M1 (also
known as PK-M or -A). The PK-M2 isoenzyme
(also known as PK-K or -C) is widely distributed
in the body and is the most important isoenzyme
of the kidney medulla, lungs, gut, fat tissue and
leukocytes. PK-M1 and -M2 arise by alternative
splicing of transcripts from the same gene [252].
PK-L (also known as PK-B) is the predominant
form in the liver; it is present only in the hepatocytes, and the other cells of the liver contain PKM. The proximal kidney tubules also contain PKL. The fourth isoenzyme, PK-R, is the only form
found in erythrocytes. Embryonal muscle initially
has PK-M2' but this is gradually replaced by PKMI' The occurrence of M2 and MI or L in the same
cell leads to the formation of MIM2 or LM2 heterotetramers [12].
The PK-L of mammalian liver is regulated
allosterically. It is activated by PEP, Fru-l,6-P2
and H+, and inhibited by ATP, OH-, alanine, phenylalanine or other amino acids; when inhibited it
shows sigmoidal PEP kinetics. Thus, apart from
the PK substrate itself, the product of the previous regulatory enzyme, PFK, is also stimulatory.
At physiological PEP concentrations, PK-L has
very low activity, but it is strongly stimulated by
only micromolar concentrations of Fru-l,6-P2.
Alanine and other glucogenic amino acids have
an inhibitory effect on PK and therefore favour
gluconeogenesis. A cAMP-dependent protein
kinase can phosphorylate PK-L on the Nterminal serine residues of the four subunits. The
phosphorylated form has a lower affinity for the
activators PEP and Fru-1.6-P2 but a higher affinity for the inhibitors ATP and alanine. Conversely, the activators can hinder the phosphorylation. Because both activators and inhibitors are present under physiological conditions,
the enzyme activity in vivo is strongly reduced
by phosphorylation; V max, however, remains
unchanged. The hormonal stimulation of gluconeogenesis involves, in particular, the cAMPdependent phosphorylation and inactivation of
the liver PK. In contrast, the PE/pyruvate substrate cycle appears to be relatively unimportant
for the regulation of glycolysis and gluconeogenesis. This substrate cycle results from the production of pyruvate from PEP by the cytosolic PK
and the simultaneous conversion of pyruvate into
PEP by the combined activities of the mitochondrial PC and the cytosolic-mitochondrial PEPCK
(see Fig. 14.1) The dephosphorylation of PK is
brought about by a non-specific protein phosphatase which is not the same as the phosphorylase
dephosphorylating enzyme. The PK-M2 and the
PK-R of the erythrocytes are similar to the liver
enzyme in their allosteric regulation; however,
the homo- and heterotropic allosteric interactions
are less marked in PK-M2 than in PK-L and PK-R
[100, 253].
In contrast to the other isoenzymes, the PK-Mt
of muscle always shows hyperbolic kinetics with
PEP as the substrate, and it is not regulated allosterically under physiological conditions. PK-M is
activated only by Fru-l,6-P2 at non-physiological,
lower magnesium concentrations or in the presence of the inhibitor phenylalanine [173, 178].
The seal Phoca hispida and other diving marine
mammals differ from the other mammals in that
their skeletal muscle and heart contain different
PK isoenzymes. The heart form corresponds to
the M-type of other species, whereas the muscle
enzyme appears to be particularly well adapted to
wide temperature fluctuations and to the alternation of long periods of anaerobic glycolysis with
short phases of fatty acid oxidation [86].
The sequence of 543 amino acids of the rat
PK-L agrees by 67-68 % with the two rat PK-M
forms, by 68 % with the PK-M1 of the chicken,
and still by 48 % with the yeast PK [111, 143, 178].
The sequences of rat PK-M1 and -M2 (531 amino
acids) are identical apart from a partial sequence
of 45 amino acids in the C-terminal domain
(amino acids 388-432). The corresponding
mRNAs are apparently produced from the primary transcript of the same gene by alternative
splicing. In the variable partial sequence, M2 is
more similar to the L type than to Mb and this
region thus appears to be responsible for the
allosteric regulation of PK [178]. PK-R is very
similar to PK-L in amino acid composition, cleavage peptide pattern and immunological characteristics [111]. The erythrocyte PKs of different
mammals show immunological cross-reactivity
but vary greatly in their kinetics, e.g. the Hill
coefficients of PEP kinetics vary under the same
conditions from a value of 0.94 in the opossum
Didelphis marsupialis, through values of 1.11 in
humans and 1.53 in rabbits to a value of 2.41 in
cattle; the opossum shows no ATP inhibition.
14 Glycolysis
PEP metabolism must be introduced; this is especially the case in many invertebrates [173].
14.7.1 Pyruvate Kinase Isoenzymes
of Mammals
Mammals and birds have four tissue-specific
pyruvate kinase isoenzymes. All the activity in
skeletal muscle and most of the activity in heart
muscle and the brain stems from PK-M1 (also
known as PK-M or -A). The PK-M2 isoenzyme
(also known as PK-K or -C) is widely distributed
in the body and is the most important isoenzyme
of the kidney medulla, lungs, gut, fat tissue and
leukocytes. PK-M1 and -M2 arise by alternative
splicing of transcripts from the same gene [252].
PK-L (also known as PK-B) is the predominant
form in the liver; it is present only in the hepatocytes, and the other cells of the liver contain PKM. The proximal kidney tubules also contain PKL. The fourth isoenzyme, PK-R, is the only form
found in erythrocytes. Embryonal muscle initially
has PK-M2' but this is gradually replaced by PKMI' The occurrence of M2 and MI or L in the same
cell leads to the formation of MIM2 or LM2 heterotetramers [12].
The PK-L of mammalian liver is regulated
allosterically. It is activated by PEP, Fru-l,6-P2
and H+, and inhibited by ATP, OH-, alanine, phenylalanine or other amino acids; when inhibited it
shows sigmoidal PEP kinetics. Thus, apart from
the PK substrate itself, the product of the previous regulatory enzyme, PFK, is also stimulatory.
At physiological PEP concentrations, PK-L has
very low activity, but it is strongly stimulated by
only micromolar concentrations of Fru-l,6-P2.
Alanine and other glucogenic amino acids have
an inhibitory effect on PK and therefore favour
gluconeogenesis. A cAMP-dependent protein
kinase can phosphorylate PK-L on the Nterminal serine residues of the four subunits. The
phosphorylated form has a lower affinity for the
activators PEP and Fru-1.6-P2 but a higher affinity for the inhibitors ATP and alanine. Conversely, the activators can hinder the phosphorylation. Because both activators and inhibitors are present under physiological conditions,
the enzyme activity in vivo is strongly reduced
by phosphorylation; V max, however, remains
unchanged. The hormonal stimulation of gluconeogenesis involves, in particular, the cAMPdependent phosphorylation and inactivation of
the liver PK. In contrast, the PE/pyruvate substrate cycle appears to be relatively unimportant
for the regulation of glycolysis and gluconeogenesis. This substrate cycle results from the production of pyruvate from PEP by the cytosolic PK
and the simultaneous conversion of pyruvate into
PEP by the combined activities of the mitochondrial PC and the cytosolic-mitochondrial PEPCK
(see Fig. 14.1) The dephosphorylation of PK is
brought about by a non-specific protein phosphatase which is not the same as the phosphorylase
dephosphorylating enzyme. The PK-M2 and the
PK-R of the erythrocytes are similar to the liver
enzyme in their allosteric regulation; however,
the homo- and heterotropic allosteric interactions
are less marked in PK-M2 than in PK-L and PK-R
[100, 253].
In contrast to the other isoenzymes, the PK-Mt
of muscle always shows hyperbolic kinetics with
PEP as the substrate, and it is not regulated allosterically under physiological conditions. PK-M is
activated only by Fru-l,6-P2 at non-physiological,
lower magnesium concentrations or in the presence of the inhibitor phenylalanine [173, 178].
The seal Phoca hispida and other diving marine
mammals differ from the other mammals in that
their skeletal muscle and heart contain different
PK isoenzymes. The heart form corresponds to
the M-type of other species, whereas the muscle
enzyme appears to be particularly well adapted to
wide temperature fluctuations and to the alternation of long periods of anaerobic glycolysis with
short phases of fatty acid oxidation [86].
The sequence of 543 amino acids of the rat
PK-L agrees by 67-68 % with the two rat PK-M
forms, by 68 % with the PK-M1 of the chicken,
and still by 48 % with the yeast PK [111, 143, 178].
The sequences of rat PK-M1 and -M2 (531 amino
acids) are identical apart from a partial sequence
of 45 amino acids in the C-terminal domain
(amino acids 388-432). The corresponding
mRNAs are apparently produced from the primary transcript of the same gene by alternative
splicing. In the variable partial sequence, M2 is
more similar to the L type than to Mb and this
region thus appears to be responsible for the
allosteric regulation of PK [178]. PK-R is very
similar to PK-L in amino acid composition, cleavage peptide pattern and immunological characteristics [111]. The erythrocyte PKs of different
mammals show immunological cross-reactivity
but vary greatly in their kinetics, e.g. the Hill
coefficients of PEP kinetics vary under the same
conditions from a value of 0.94 in the opossum
Didelphis marsupialis, through values of 1.11 in
humans and 1.53 in rabbits to a value of 2.41 in
cattle; the opossum shows no ATP inhibition.