very similar in its spatial structure to the
nucleotide-binding domains of other dehydrogenases (Rossmann fold), and a catalytic domain.
Yeast GAPDH shows positive cooperativity,
whereas the animal GAPDHs are negatively
cooperative, with Hill coefficients of around 0.8.
According to investigations of the muscle
GAPDHs of the sturgeon species Huso huso and
Acipenser transmontanus, this results from the
fact that the first two NAD+ are bound to nonneighbouring binding sites of high affinity, and
the next two are bound to neighbouring interacting sites [266].
The amino acid sequences of the GAPDHs
from Drosophila, lobster, chicken, pig, rat and
humans have 63 % identical amino acids; the
GAPDH of Caenorhabditis is 67 % similar to that
of Drosophila and the chicken. The GAPDHs of
bakers' yeast and two bacterial species have 32 %
of amino acids in common with the animal enzymes [48, 159,257,283]. Thus, GAPDH is a protein with a relatively low rate of evolution (see
Table 4.12, p. 161). In agreement with this conclusion is the fact that the GAPDH subunits of rabbit and yeast can form functional heterotetramers. At least two-thirds of the amino acids that
are invariant in all organisms are found in the
catalytic domain; the nucleotide-binding domain
is highly conserved in its spatial structure but not
in its amino acid sequence [48, 257].
The chicken GAPDH gene has 11 introns, 3 of
which lie on the boundaries between the
nucleotide-binding domain, the catalytic domain
and the helical tail region. The human gene has
only eight introns, and the equivalent of chicken
exons VIII-XI are fused together; the remaining
introns occupy similar positions to those in the
chicken gene [55]. The GAPDH gene of Caenorhabditis elegans has only two introns [283].
Although only one enzyme protein is produced,
the human genome contains 100 copies of the
gene, and in the rat there are as many as 300-400
copies; many pseudo genes are found without
introns and with many nucleotide substitutions.
The presence of transcription products with differing sequences suggests that some of these
pseudogenes are transcribed [210]. There is
apparently only one functional human gene, and
isoenzymes are in general not found in the vertebrates. In contrast, the nematode Caenorhabditis
elegans has two tissue-specific isoenzymes
encoded by two pairs of genes. The almost identical genes, gdp-1 and gdp-4, give rise to GAPDH1, which is found at low activity in all cells; genes
gdp-2 and gdp.3 produce the more active, muscle14.7 Pyruvate Kinases
529
specific GAPDH-2. The two isoenzymes differ
mostly in a cluster of amino acids at the Nterminus [106,282]. Drosophila also has two
GAPDH isoenzymes encoded by different genes
[247], and Trypanosoma brucei possesses two
GAPDH isoenzymes with different intracellular
localizations: one is found in the glycosomes and
makes up 80 % of the total activity, whilst the
other is soluble in the cytosol. The glycosomal
enzyme (358 amino acids) has only 55 % of its
sequence in common with the cytosolic enzyme
(330 amino acids), and is much more basic with a
charge excess of + 11 compared with +2 for the
cytosolic enzyme. The glycosomal enzyme is
about 4 kDa larger than the cytosolic enzyme as
the result of seven insertions of one to eight
amino acids and a C-terminal extension of seven
amino acids [160].
14.7 Pyruvate Kinases
The pyruvate kinases (PK) of the vertebrates and
invertebrates are usually tetramers of about 60kDa subunits. The liver of the rat and the flounder Platichthys flesus also contain higher polymers of several megadaltons [36, 216, 245].
Tissue-specific isoenzymes of PK are found in all
vertebrates (with the exception of the cartilaginous fish), and these differ markedly in their structural and kinetic-regulatory properties. All PKs
are apparently homologous; those from bakers'
yeast and the chicken agree at 55 % of positions
[142].
The PKs constitute the last potential point of
regulation of glycolysis; however, there are radical differences in kinetic-regulatory properties
between the tissue-specific isoenzymes of the vertebrates and the PKs of various invertebrates.
The necessity for PK regulation in certain metabolic situations is obvious. During gluconeogenesis, for example, the PK reaction cannot be
reversed for thermodynamic reasons; the conversion of pyruvate to phosphoenolpyruvate (PEP)
occurs rather by the enzymes pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK). However, even in tissues with a
relatively high capacity for gluconeogenesis, e.g.
mammalian liver, the maximal PK activity is significantly higher than the PC and PEPCK activities. Thus, PK must be down-regulated during
gluconeogenesis. The inhibition of PK is also necessary during hypoxia, when pyruvate is not
reduced to lactate or opines and other routes for
nucleotide-binding domains of other dehydrogenases (Rossmann fold), and a catalytic domain.
Yeast GAPDH shows positive cooperativity,
whereas the animal GAPDHs are negatively
cooperative, with Hill coefficients of around 0.8.
According to investigations of the muscle
GAPDHs of the sturgeon species Huso huso and
Acipenser transmontanus, this results from the
fact that the first two NAD+ are bound to nonneighbouring binding sites of high affinity, and
the next two are bound to neighbouring interacting sites [266].
The amino acid sequences of the GAPDHs
from Drosophila, lobster, chicken, pig, rat and
humans have 63 % identical amino acids; the
GAPDH of Caenorhabditis is 67 % similar to that
of Drosophila and the chicken. The GAPDHs of
bakers' yeast and two bacterial species have 32 %
of amino acids in common with the animal enzymes [48, 159,257,283]. Thus, GAPDH is a protein with a relatively low rate of evolution (see
Table 4.12, p. 161). In agreement with this conclusion is the fact that the GAPDH subunits of rabbit and yeast can form functional heterotetramers. At least two-thirds of the amino acids that
are invariant in all organisms are found in the
catalytic domain; the nucleotide-binding domain
is highly conserved in its spatial structure but not
in its amino acid sequence [48, 257].
The chicken GAPDH gene has 11 introns, 3 of
which lie on the boundaries between the
nucleotide-binding domain, the catalytic domain
and the helical tail region. The human gene has
only eight introns, and the equivalent of chicken
exons VIII-XI are fused together; the remaining
introns occupy similar positions to those in the
chicken gene [55]. The GAPDH gene of Caenorhabditis elegans has only two introns [283].
Although only one enzyme protein is produced,
the human genome contains 100 copies of the
gene, and in the rat there are as many as 300-400
copies; many pseudo genes are found without
introns and with many nucleotide substitutions.
The presence of transcription products with differing sequences suggests that some of these
pseudogenes are transcribed [210]. There is
apparently only one functional human gene, and
isoenzymes are in general not found in the vertebrates. In contrast, the nematode Caenorhabditis
elegans has two tissue-specific isoenzymes
encoded by two pairs of genes. The almost identical genes, gdp-1 and gdp-4, give rise to GAPDH1, which is found at low activity in all cells; genes
gdp-2 and gdp.3 produce the more active, muscle14.7 Pyruvate Kinases
529
specific GAPDH-2. The two isoenzymes differ
mostly in a cluster of amino acids at the Nterminus [106,282]. Drosophila also has two
GAPDH isoenzymes encoded by different genes
[247], and Trypanosoma brucei possesses two
GAPDH isoenzymes with different intracellular
localizations: one is found in the glycosomes and
makes up 80 % of the total activity, whilst the
other is soluble in the cytosol. The glycosomal
enzyme (358 amino acids) has only 55 % of its
sequence in common with the cytosolic enzyme
(330 amino acids), and is much more basic with a
charge excess of + 11 compared with +2 for the
cytosolic enzyme. The glycosomal enzyme is
about 4 kDa larger than the cytosolic enzyme as
the result of seven insertions of one to eight
amino acids and a C-terminal extension of seven
amino acids [160].
14.7 Pyruvate Kinases
The pyruvate kinases (PK) of the vertebrates and
invertebrates are usually tetramers of about 60kDa subunits. The liver of the rat and the flounder Platichthys flesus also contain higher polymers of several megadaltons [36, 216, 245].
Tissue-specific isoenzymes of PK are found in all
vertebrates (with the exception of the cartilaginous fish), and these differ markedly in their structural and kinetic-regulatory properties. All PKs
are apparently homologous; those from bakers'
yeast and the chicken agree at 55 % of positions
[142].
The PKs constitute the last potential point of
regulation of glycolysis; however, there are radical differences in kinetic-regulatory properties
between the tissue-specific isoenzymes of the vertebrates and the PKs of various invertebrates.
The necessity for PK regulation in certain metabolic situations is obvious. During gluconeogenesis, for example, the PK reaction cannot be
reversed for thermodynamic reasons; the conversion of pyruvate to phosphoenolpyruvate (PEP)
occurs rather by the enzymes pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK). However, even in tissues with a
relatively high capacity for gluconeogenesis, e.g.
mammalian liver, the maximal PK activity is significantly higher than the PC and PEPCK activities. Thus, PK must be down-regulated during
gluconeogenesis. The inhibition of PK is also necessary during hypoxia, when pyruvate is not
reduced to lactate or opines and other routes for