The reaction of the pyruvate dehydrogenase complex is shown in Figure 6.2; the reactions of the 2-oxoglutarate and branched-chain oxo-acid dehydrogenase complexes follow the same sequence, and the multienzyme complexes are similar.
Thiamin binds the oxo-acid substrate, decarboxylating it to an active aldehyde intermediate. This is then transferred to enzyme-bound lipoamide, reducing the disulfide bridge of the lipoamide and forming a thioester. The resultant acyl group is transferred to coenzyme A (CoA), and the dithiol lipoamide is reoxidized by NAD+.
The multienzyme complexes are self-assembling and will reassemble to an active complex after resolution of the individual enzymes. The core enzyme of the complex is the dihydrolipoyl acyltransferase (E2); the oxo-acid dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) subunits form noncovalent bonds to this central catalytic unit.
The pyruvate dehydrogenase E1 subunit is a tetramer of two classes of sub-units. The a-subunit catalyzes the decarboxylation of pyruvate to hydroxy-ethylthiamine diphosphate and the ^-subunit catalyzes the reductive acy-lation of the lipoamide prosthetic group of the acetyltransferase subunit. In the complete multienzyme complex, there are 20 (kidney) or 30 (heart) dehydrogenase tetramers, 60 acetyltransferase subunits, 10 (kidney) or 12 (heart) dihydrolipoyl dehydrogenase subunits, and 5 each of the kinase and phos-phatase regulatory subunits.
18.104.22.168 Regulation of Pyruvate Dehydrogenase Activity Pyruvate dehy-drogenase is the key enzyme that commits pyruvate (and hence the products of carbohydrate metabolism) to complete oxidation (via the tricarboxylic acid cycle) or lipogenesis. It is subject to regulation by both product inhibition and a phosphorylation/dephosphorylation mechanism. Acetyl CoA and NADH are both inhibitors, competing with coenzyme A and NAD+.
There are four isoenzymes of the kinase. Pyruvate dehydrogenase is inhibited by phosphorylation of three serine residues on the E1a subunit; this reduces the affinity for thiamin diphosphate 12-fold. Kinase 1 phosphorylates all three sites, whereas the other isoenzymes phosphorylate only two sites, and kinase 2 may catalyze only partial phosphorylation of one of these sites. Binding of thiamin diphosphate to E1a decreases the phosphorylation of the enzyme by the kinases. Pyruvate dehydrogenase is reactivated by dephospho-rylation, catalyzed by two phosphatases, which have different activity on the enzyme depending on which kinase has catalyzed the phosphorylation. This means that regulation of pyruvate dehydrogenase activity will differ in different tissues, depending on the tissue-specific expression of the kinases and phosphatases (Kolobova et al., 2001; Korotchkina and Patel, 2001a, 2001b).
The kinases are inhibited by pyruvate and adenosine disphosphate (ADP), and the phosphatases are activated by calcium ions. There is normally a constant process of phosphorylation and dephosphorylation of the enzyme, so that it is very sensitive to changes in intracellular free calcium and the adeno-sine triphosphate (ATP):ADP ratio.
Pyruvate dehydrogenase kinases are induced by glucocorticoid hormones and long-chain fatty acids (acting via the peroxisome proliferation-activated receptor). This effect is antagonized by insulin, thus reducing pyruvate dehydrogenase activity in starvation and suggesting abnormalities of regulation of pyruvate metabolism in diabetes mellitus (Sugden et al., 2001a, 2001b; Huang etal., 2002).
In addition to its cofactor role, thiamin diphosphate, together with calcium or other divalent cations, activates pyruvate dehydrogenase by binding to a regulatory site and reducing the Km for pyruvate (Czerniecki and Czygier, 2001).
22.214.171.124 Thiamin-Responsive Pyruvate Dehydrogenase Deficiency Genetic deficiency of pyruvate dehydrogenase E1a (which is on the X chromosome) leads to potentially fatal lactic acidosis, with psychomotor retardation, central nervous system damage, atrophy of muscle fibers and ataxia, and developmental delay. At least some cases respond to the administration of high doses (20 to 3,000 mg per day) of thiamin. In those cases where the enzyme has been studied, there is a considerable increase in the Km of the enzyme for thiamin diphosphate. Female carriers of this X-linked disease are affected to a variable extent, depending on the X-chromosome inactivation pattern in different tissues (Robinson et al., 1996).
126.96.36.199 2-Oxoglutarate Dehydrogenase and the 7-Aminobutyric Acid (GABA) Shunt 2-Oxoglutarate dehydrogenase catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl CoA in the citric acid cycle. There is considerably less impairment of citric acid cycle activity (and hence ATP formation) in thiamin deficiency than might be expected, and unlike pyru-vate, 2-oxoglutarate does not accumulate in the brains of thiamin-deficient animals. There is a significant reduction in brain 2-oxoglutarate (Butterworth andHeroux, 1989).
As shown in Figure 6.3, the formation and catabolism of the neurotrans-mitter y-aminobutyric acid (GABA) provides an alternative to 2-oxoglutarate dehydrogenase - the so-called GABA shunt. 2-Oxoglutarate is aminated to glutamate by the reaction of either glutamate dehydrogenase or a variety of transaminases; glutamate then undergoes decarboxylation to GABA. GABA is inactivated by transamination (in which 2-oxoglutarate is the amino acceptor), yielding succinic semialdehyde, which is oxidized to succinate, an intermediate in the tricarboxylic acid cycle.
Glutamate decarboxylase and GABA aminotransferase are found in regions of the central nervous system other than those in which GABA has a
neurotransmitter role, and also in nonneuronal tissues, including the liver and kidneys. In a number of studies, there is evidence of a decrease in the total brain concentration of GABA in thiamin deficiency, but studies with [14C]glutamate show that there is a considerable increase in the rate of GABA turnover, and suggest that the GABA shunt may be a significant alternative to 2-oxoglutarate dehydrogenase in energy-yielding metabolism in thiamin deficiency, permitting continued tricarboxylic acid cycle activity despite the impairment of 2-oxoglutarate dehydrogenase (Page et al., 1989).
188.8.131.52 Branched-Chain Oxo-acid Decarboxylase and Maple Syrup Urine Disease The third oxo-acid dehydrogenase catalyzes the oxidative decarboxylation of the branched-chain oxo-acids that arise from the transamination of the branched-chain amino acids, leucine, isoleucine, and valine. It has a similar subunit composition to pyruvate and 2-oxoglutarate dehydrogenases, and the E3 subunit (dihydrolipoyl dehydrogenase) is the same protein as in the other two multienzyme complexes. Genetic lack of this enzyme causes maple syrup urine disease, so-called because the branched-chain oxo-acids that are excreted in the urine have a smell reminiscent of maple syrup.
Like the other thiamin diphosphate-dependent dehydrogenases, branched-chain oxo-acid dehydrogenase is regulated by phosphorylation and dephosphorylation, and the proportion of the enzyme in the active (de-phosphorylated) state is high in the liver, low in skeletal muscle, and intermediate in the kidneys and heart. In most tissues, the activity of the dehy-drogenase is considerably lower than that of branched-chain amino acid aminotransferase, and regulation of the dehydrogenase is therefore important for control of branched-chain amino acid metabolism, and overall amino acid and nitrogen metabolism (Lombardo et al., 1999; Harris et al., 2001; Obayashi etal., 2001).
Various mutations affecting either the E1 or the E2 subunit of the dehydrogenase are involved in different forms of maple syrup urine disease. Acute infantile disease is caused by near complete lack of activity of the enzyme. The intermittent form of the disease is associated with marginally adequate residual activity of the enzyme that is able to cope with the branched-chain oxo-acids arising from the metabolism of modest amounts of branched-chain amino acids, but not relatively large amounts.
Some patients with maple syrup urine disease respond to high doses (10 to 1,000 mg per day) of thiamin; in some patients, the defect is clearly in the E1a subunit, which has a Km for thiamin diphosphate 16-fold higher than normal. But in other thiamin-responsive cases, the mutation is in the E2 subunit, suggesting that assembly of the active multienzyme complex affects the affinity of the thiamin diphosphate binding site of the E1a subunit.
In vitro, thiamin diphosphate inhibits the kinase that phosphorylates and inactivates branched-chain oxo-acid dehydrogenase, and might be expected to increase the activity of the enzyme in tissues, thus offering an alternative mechanism for thiamin-responsive maple syrup urine disease. However, this seems not to be relevant in vivo, possibly because tissue concentrations of thiamin diphosphate do not rise high enough to affect the activity of the kinase. In thiamin-deficient animals, there is an increase in the total liver content of branched-chain oxo-acid dehydrogenase (suggesting induction of the enzyme) and an increase in the proportion in the dephosphorylated active form. Feeding animals with very high levels of thiamin results in a decrease in the total liver content of the dehydrogenase and a decrease in the proportion in the dephosphorylated state (Blair et al., 1999).
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