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Subjective Questions- Fate of pyruvate (Solved)
Answer- The sequence of reactions from glucose to pyruvate is similar in most organisms and most types of cells. In contrast, the fate of pyruvate is variable. Three reactions of pyruvate are of prime importance: conversion into ethanol, lactic acid, or carbon dioxide
1. Ethanol is formed from pyruvate in yeast and several other microorganisms. The first step is the decarboxylation of pyruvate. This reaction is catalyzed by pyruvate decarboxylase, which requires the coenzyme thiamine pyrophosphate.
This coenzyme, derived from the vitamin thiamine (B1), also participates in reactions catalyzed by other enzymes. The second step is the reduction of acetaldehyde to ethanol by NADH, in a reaction catalyzed by alcohol dehydrogenase.This process regenerates NAD+.
Figure-1- showing the conversion of pyruvate to Ethanol
The net result of this anaerobic process is:
The conversion of glucose into ethanol is an example of alcoholic fermentation. NADH generated by the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of acetaldehyde to ethanol. Thus, there is no net oxidation-reduction in the conversion of glucose into ethanol (Figure-1). The ethanol formed in alcoholic fermentation provides a key ingredient for brewing and wine making.
2. Lactate is formed from pyruvate in a variety of microorganisms in a process called lactic acid fermentation. The reaction also takes place in the cells of higher organisms when the amount of oxygen is limiting, as in muscle during intense activity. The reduction of pyruvate by NADH to form lactate is catalyzed by lactate dehydrogenase.
Figure-2- showing the conversion of pyruvate to Lactate
The overall reaction in the conversion of glucose into lactate is:
As in alcoholic fermentation, there is no net oxidation-reduction. The NADH formed in the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of pyruvate. The regeneration of NAD + in the reduction of pyruvate to lactateor ethanol sustains the continued operation of glycolysis under anaerobic conditions.
3. Acetyl co A – Only a fraction of the energy of glucose is released in its anaerobic conversion into ethanol or lactate. Much more energy can be extracted aerobically by means of the citric acid cycle and the electron-transport chain. The entry point to this oxidative pathway is acetyl coenzyme A (acetyl CoA), which is formed inside mitochondria by the oxidative decarboxylation of pyruvate.
The NAD+ required for this reaction and for the oxidation of glyceraldehyde 3-phosphate is regenerated when NADH ultimately transfers its electrons to O2 through the electron-transport chain in mitochondria. The reaction is catalyzed by a multi enzyme complex called Pyruvate dehydrogenase complex.
4. Oxaloacetate- Pyruvate can be converted to oxaloacetate. Mitochondrial pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate, an ATP-requiring reaction in which the vitamin biotin is the coenzyme. Biotin binds CO2 from bicarbonate as carboxybiotin prior to the addition of the CO2 to pyruvate.
Figure –3- showing the conversion of pyruvate to oxaloacetate
The Oxaloacetate can be subsequently used for the synthesis of Aspartate, phosphoenol pyruvate or be utilized in the TCA cycle depending upon the need of the cell.
5. Alanine- Pyruvate can be transaminated to form Alanine as per need.
Figure-4- showing the conversion of Pyruvate to Alanine by transamination
This reaction is important for the catabolism and synthesis of non-essential amino acids
6. Malate- Pyruvate can be directly converted to oxaloacetate or it is first carboxylated to malate and then decarboxylated to from oxaloacetate (Figure-5). These two reactions are called CO2 filling up reactions or Anaplerotic reactions. They provide oxaloacetate when there is sudden influx of Acetyl co A in the TCA cycle.
Figure-5- showing the formation of Oxalo acetate from Pyruvate.
Thus Pyruvate can be metabolized through several pathways as per availability of O2 or requirement of the cell for a specific metabolite.
Q.- 2-Give a brief account of Pyruvate dehydrogenase complex. How is this complex regulated?
Answer- Under anaerobic conditions, pyruvate is converted into lactic acid or ethanol, depending on the organism. Under aerobic conditions, the pyruvate is transported into mitochondria in exchange for OH- by the pyruvate carrier, an antiporter. In the mitochondrial matrix, pyruvate is oxidatively decarboxylated by the pyruvate dehydrogenase complex to form acetyl CoA.
This irreversible reaction is the link between glycolysis and the citric acid cycle. In the preparation of the glucose derivative pyruvate for the citric acid cycle, an oxidative decarboxylation takes place and high transfer- potential electrons in the form of NADH are captured.
Pyruvate dehydrogenase complex
The pyruvate dehydrogenase complex is a large, highly integrated complex of three kinds of enzymes; Pyruvate dehydrogenase, dihydrolipoyl transacetylase and Dihydrolipoyl dehydrogenase. At least two additional enzymes regulate the activity of the complex and five coenzymes: thiamine pyrophosphate (TPP), lipoic acid, CoASH, FAD and NAD+ participate in the overall reaction. The pyruvate dehydrogenase complex consists of a number of polypeptide chains of each of the three component enzymes, and the intermediates do not dissociate, but remain bound to the enzymes.
Pyruvate dehydrogenase is a member of a family of homologous complexes that includes the citric acid cycle enzyme, alpha ketoglutarate dehydrogenase and for branched-chain amino acids, alpha-ketoacid dehydrogenase,These complexes are large, with molecular masses ranging from 4 to 10 million daltons.
The conversion of pyruvate into acetyl Co A consists of three steps: decarboxylation, oxidation, and transfer of the resultant acetyl group to CoA.
Figure-6 showing the processes involved in the conversion of pyruvate to Acetyl co A
These steps must be coupled to preserve the free energy derived from the decarboxylation step to drive the formation of NADH and acetyl CoA.
1) Pyruvate is decarboxylated by the pyruvate dehydrogenase component of the enzyme complex to a hydroxyethyl derivative of the thiazole ring of enzyme-bound thiamin diphosphate,
2) Hydroxy ethyl TPP (Acyl TPP) reacts with oxidized lipoamide, the prosthetic group of dihydrolipoyl transacetylase, to form acetyl lipoamide. In thiamin (vitamin B1 ) deficiency, glucose metabolism is impaired, and there is significant (and potentially life-threatening) lactic and pyruvic acidosis.
3)Acetyl lipoamide reacts with coenzyme A to form acetyl-CoA and reduced lipoamide.
4)The reaction is completed when the reduced lipoamide is reoxidized by a flavoprotein, dihydrolipoyl dehydrogenase, containing FAD.
5) Finally, the reduced flavoprotein is oxidized by NAD+, which in turn transfers reducing equivalents to the respiratory chain.
Each NADH yields 3 ATPS in the electron transport chain. Since from each Glucose two pyruvate molecules are produced, thus the net energy yield at this step is 6 ATPs.
Figure-7- Showing the reactions of pyruvate dehydrogenase complex. During the oxidation of pyruvate to CO2 by pyruvate dehydrogenase the electrons flow from pyruvate to the lipoamide moiety of dihydrolipoyl transacetylase then to the FAD cofactor of dihydrolipoyl dehydrogenase and finally to reduction of NAD+ to NADH. The acetyl group is linked to coenzyme A (CoASH) in a high energy thioester bond. The acetyl-CoA then enters the TCA cycle for complete oxidation to CO2 and H2O.
Regulation of PDH complex
The reactions of the PDH complex serve to interconnect the metabolic pathways of glycolysis, gluconeogenesis and fatty acid synthesis to the TCA cycle. As a consequence, the activity of the PDH complex is highly regulated by a variety of allosteric effectors and by covalent modification.
Pyruvate dehydrogenase is inhibited by its products, acetyl-CoA and NADH.
It is also regulated by phosphorylation of three serine residues on the pyruvate dehydrogenase component of the multienzyme complex by a specific PDH kinase, resulting in decreased activity, and by dephosphorylation by a PDH phosphatase that causes an increase in activity
NADH and acetyl-CoA are powerful positive effectors on PDH kinase, the enzyme that inactivates PDH by converting it to the phosphorylated PDH-b form. The kinase is activated by increases in the [ATP]/[ADP], [acetyl-CoA]/[CoASH], and [NADH]/[NAD+] ratios. Since NADH and acetyl-CoA accumulate when the cell energy charge is high, it is not surprising that high ATP levels also up-regulate PDH kinase activity, reinforcing down-regulation of PDH activity in energy-rich cells. Note, however, that pyruvate is a potent negative effector on PDH kinase, with the result that when pyruvate levels rise, PDH-a will be favored even with high levels of NADH and acetyl-CoA.
Concentrations of pyruvate which maintain PDH in the active form (PDH-a) are sufficiently high so that, in energy-rich cells, the allosterically down-regulated, high Km form of PDH is nonetheless capable of converting pyruvate to acetyl-CoA. With large amounts of pyruvate in cells having high energy charge and high NADH, pyruvate carbon will be directed to the 2 main storage forms of carbon (glycogen via gluconeogenesis and fat production via fatty acid synthesis) where acetyl-CoA is the principal carbon donor.
Figure-8- showing the regulation of PDH complex
Although the regulation of PDH-b phosphatase is not well understood, it is likely to be regulated to maximize pyruvate oxidation under energy-poor conditions and to minimize PDH activity under energy-rich conditions. It is known that Mg2+ and Ca2+ activate the enzyme
Thus, pyruvate dehydrogenase, and therefore glycolysis, is inhibited both when there is adequate ATP (and reduced coenzymes for ATP formation) available, and also when fatty acids are being oxidized. In fasting, when free fatty acid concentrations increase, there is a decrease in the proportion of the enzyme in the active form, leading to a sparing of carbohydrate. In adipose tissue, glucose provides acetyl-CoA for lipogenesis, the enzyme is activated in response to insulin and in cardiac muscle PDH activity is increased by catecholamines.
Q.3-What would be the consequences of Pyruvate dehydrogenase complex deficiency?
Answer– Pyruvate dehydrogenase complex deficiency (PDCD) is a rare disorder of carbohydrate metabolism caused by a deficiency of one or more enzymes in the pyruvate dehydrogenase complex. Dysfunctions in all 3 substrate-processing enzymes, regulatory proteins and thiamine dependence of the E1 alpha enzyme, have been described; however, dysfunction of the E1 alpha enzyme subunit is most common.
The age of onset and severity of disease depends on the activity level of the PDC enzymes. Individuals with PDCD beginning prenatally or in infancy usually die in early childhood. Those who develop PDCD later in childhood may have mental retardation and other neurological symptoms and usually survive into adulthood.
The following features are characteristic of this disease-
1) Energy Deficit-A deficiency in this enzymatic complex limits the production of citrate. Because citrate is the first substrate in the citric acid cycle, the cycle cannot proceed. Alternate metabolic pathways are stimulated in an attempt to produce acetyl-CoA; however, an energy deficit remains, especially in the CNS. The magnitude of the energy deficit depends on the residual activity of the enzyme.
2) Neurological deficit– Severe enzyme deficiencies may lead to congenital brain malformation because of a lack of energy during neural development. Underlying neuropathology is not usually observed in individuals whose onset of pyruvate dehydrogenase complex deficiency is in childhood.
The signs of poor neurological development or degenerative lesions are -Poor acquisition or loss of motor milestones, poor muscle tone, new onset seizures, and periods of incoordination (i.e., ataxia) abnormal eye movements, poor response to visual stimuli, mental delay, psychomotor delays and growth retardation
High blood lactate and pyruvate levels with or without lactic acidemia suggest an inborn error of metabolism at the mitochondrial level.
Cofactor supplementation with thiamine, carnitine, and Lipoic acid is the standard of care. Ketogenic diets (with restricted carbohydrate intake) have been used to control lactic acidosis with minimal success.
Correction of acidosis does not reverse all the symptoms. CNS damage is common and limits recovery of normal function.