CHAPTER 27: METABOLIC FATES OF PYRUVATE

Chapter 27: Metabolic Fates of Pyruvate
Approximately 90% of available metabolic energy that was present in the original glucose molecule remains in the two moles of pyruvate produced from it through anaerobic glycolysis. If anaerobic conditions prevail and pyruvate cannot be oxidized to CO2 and H2O in mitochondria, it may be converted to lactate so that NAD+ is regenerated for continued operation of glycolysis and ATP formation in the cytoplasm. Under anaerobic conditions, this is quantitatively the most important process for reoxidation of NADH in the cytoplasm of vertebrate cells, and the enzyme catalyzing this reaction is lactate dehydrogenase (LDH, #22). Other effective means of regenerating NAD+ in the cytoplasm are to convert pyruvate (through an amino transferase reaction) to alanine, and, under aerobic conditions, to operate the glycerol 3-phosphate and malate shuttles.
Tissues damaged by disease or injury typically release their intracellular enzymes into blood, where subsequent detection and measurement of their activities can provide meaningful information regarding which tissues are most affected. Lactate dehydrogenase exists in the body as several separate isozymes, and the pattern of LDH isozymes present in blood is particularly useful in distinguishing, for example, between myocardial infarction and liver disease (such as infective hepatitis). Two different genes are known to code for LDH. One codes for the M or muscle form, and the other for the H or heart form. There are four subunits in LDH, thus giving five possible isozymes: M4, M3H, M2H2, MH3, and H4. Skeletal muscle contains some of all five isozymes (but predominantly M4), and heart has predominantly H4. The H4 isozyme is strongly inhibited by pyruvate; however, the M4 species is not. During periods of exercise when skeletal muscle is producing lactate, heart muscle removes it from the circulation forming pyruvate, which can then enter its numerous mitochondria. LDH isozymes are also present in the liver, kidneys and RBCs, and relatively little tissue injury or hemolysis can result in pronounced circulatory activity since tissue activity is generally high.
The reaction that interconverts pyruvate with alanine (Ala, reaction #23), is catalyzed by alanine aminotransferase (ALT). The carbon skeletons of pyruvate and alanine differ by only the substitution of an a-amino group for the carbonyl oxygen. This reaction is reversible, and usually operates in the direction of alanine —> pyruvate in liver (during gluconeogenesis), and in the direction of pyruvate —> alanine in muscle (particularly during exercise). Primary functions for alanine are incorporation into proteins and participation in transamination. A large amount of ammonia (NH3) is transported from muscle and other peripheral tissues to liver in the form of alanine. This process uses pyruvate produced by glycolysis to accept nitrogen from (branched-chain) amino acids in the formation of alanine, which is converted back to pyruvate in the liver where it can then participate in gluconeogenesis. Since the liver supplies glucose to other tissues, pyruvate and alanine constitute a shuttle mechanism for carrying nitrogen to the liver to be reutilized or converted to urea. Other glucogenic amino acids that can be converted to pyruvate in the liver include tryptophan, glycine, serine, cysteine, and threonine.
Another enzyme that can give rise to pyruvate in the cytoplasm is malic enzyme (#24). This enzyme is a decarboxylating enzyme that serves as an additional source of NADPH for lipogenesis. Although there is little malic enzyme activity in ruminant animals, other mammals that utilize glucose for lipogenesis (by way of citrate), are thought to rely heavily on transferring reducing equivalents from extramitochondrial NADH to NADP through the combined actions of malate dehydrogenase and malic enzyme, particularly in liver cells. The pyruvate that results from this reaction sequence is then available to reenter mitochondria for conversion to oxaloacetate or acetyl-CoA, or be converted to alanine via ALT (above). In addition to cytoplasmic conversion of malate to pyruvate, it should be noted that malate can also be shuttled back into mitochondria (usually in exchange with either citrate or a-ketoglutarate), or be converted to oxaloacetate.
Mitochondrial formation of acetyl-CoA from pyruvate is an important irreversible step in animal cells, because they are unable to further convert the acetyl-CoA to glucose. The enzyme that catalyzes this oxidative decarboxylation reaction is pyruvate dehydrogenase (PDH, #25). The acetyl-CoA generated is now committed to two principal fates:

Oxidation to CO2 and H2O via the TCA cycle (with generation of ATP through oxidative phosphorylation), or
Incorporation into other compounds (e.g., acetylcholine (an important neurotransmitter), ketone bodies, or citrate. Once citrate is formed it can either be oxidized in the TCA cycle, or it can diffuse into the cytoplasm where it becomes available for incorporation into various lipids (e.g., fatty acids and steroids).

The activity of PDH is stringently regulated. An increase in the mitochondrial NADH/NAD+, ATP/ADP, or GTP/GDP concentration ratios will inhibit this enzyme, as will a buildup of acetyl-CoA, whereas presence of insulin and an increased pyruvate concentration has a stimulatory effect. Reduced PDH activity occurs through phosphorylation of a serine residue, whereas activation occurs secondary to an increase in phosphatase activity (and consequent dephosphorylation of PDH). Thus, PDH activity is reduced when the mitochondrial energy level is high and biosynthetic intermediates are abundant (for example during fatty acid b-oxidation), and it is increased when glucose is being funneled into pyruvate and the insulin levels are elevated. Arsenite or mercuric ions inhibit PDH, as does a dietary deficiency of the B-complex vitamins, thiamin or niacin (a source of NAD+). This reaction, its cofactor requirements and metabolic inhibitors, is similar to the oxidative decarboxylation reaction of the TCA cycle that is catalyzed by a-ketoglutarate dehydrogenase.

A PDH deficiency could have serious consequences in those tissues that depend largely on complete aerobic glucose oxidation for ATP generation. This is the case for muscle, kidney, brain and peripheral nervous tissue, and there is evidence that some cases of spino-cerebellar ataxia may be due to PDH deficiency.

Pyruvate carboxylase, a zinc-containing enzyme, tags a CO2 onto pyruvate, so that oxaloacetic acid (OAA) is formed (reaction #26). To drive this reaction energetically, one ATP is needed. Furthermore, Mg++ and Mn++ are required, and the B vitamin biotin is involved as a shuttler for CO2. Pyruvate carboxylase activity is especially high in the liver and kidneys, main sites for gluconeogenesis. By producing OAA, this enzyme helps to replenish an important TCA intermediate, and also opens the way for gluconeogenesis from pyruvate and compounds that are converted to pyruvate (such as lactate and various amino acids, see the dicarboxylic acid (DCA) shuttle). A high level of acetyl-CoA inside mitochondria is required for optimal activity of pyruvate carboxylase. Acetyl-CoA serves as an allosteric activator of this enzyme, even though acetyl-CoA itself does not partake in the reaction. The mitochondrial level of acetyl-CoA starts to rise when fatty acids are broken down to fill a demand for energy, and this rise causes pyruvate carboxylase to replenish the TCA cycle (by generating OAA from pyruvate), so that acetyl-CoA can be combusted through citrate formation (note: a six-carbon citrate is formed through the condensation of a four carbon OAA, and a two-carbon acetyl-CoA). This is particularly important in exercising aerobic muscle tissue, where about 66% of the energy demand is met through fatty acid oxidation (and thus acetyl-CoA generation), and 33% through glucose oxidation (and thus OAA generation). Thus, not only is pyruvate carboxylase important in hepatic and renal gluconeogenesis, but it also plays an important role in maintaining appropriate TCA cycle intermediates in exercising muscle and other tissue. These intermediates need to be replenished because they are consumed in some biosynthetic reactions, such as heme biosynthesis. This action of pyruvate carboxylase is commonly referred to anaplerotic, meaning "to fill up".

SUMMARY

Chapter 27 discusses the metabolic fates of pyruvate. Under anaerobic conditions, pyruvate can be converted to lactate to regenerate NAD+ for glycolysis. Lactate dehydrogenase is the enzyme responsible for this reaction. The pattern of lactate dehydrogenase isozymes in the blood can be used to distinguish between different diseases. Pyruvate can also be converted to alanine through an amino transferase reaction, which is reversible and operates in different directions in the liver and muscle. Alanine serves as a shuttle mechanism for carrying nitrogen to the liver. Pyruvate can also be generated from malate through malic enzyme, which is important for lipogenesis. In the mitochondria, pyruvate is converted to acetyl-CoA by pyruvate dehydrogenase. Acetyl-CoA can be oxidized in the TCA cycle or incorporated into other compounds. The activity of pyruvate dehydrogenase is regulated by various factors. A deficiency in pyruvate dehydrogenase can have serious consequences in tissues that rely on aerobic glucose oxidation. Pyruvate carboxylase tags a CO2 onto pyruvate to form oxaloacetic acid, which is important for gluconeogenesis and replenishing TCA cycle intermediates. Acetyl-CoA serves as an allosteric activator for pyruvate carboxylase. Overall, the metabolic fates of pyruvate play crucial roles in energy production and various metabolic pathways.

OUTLINE

Chapter 27: Metabolic Fates of Pyruvate

I. Anaerobic Conditions and Pyruvate Metabolism

A. Conversion of pyruvate to lactate by lactate dehydrogenase (LDH)

B. Other means of regenerating NAD+ in the cytoplasm

1. Conversion of pyruvate to alanine by alanine aminotransferase (ALT)

2. Operation of the glycerol 3-phosphate and malate shuttles

II. LDH Isozymes and Tissue Damage

A. LDH exists as several separate isozymes

B. Pattern of LDH isozymes in blood can distinguish between different diseases

C. LDH isozymes present in skeletal muscle, heart, liver, kidneys, and RBCs

III. Alanine Metabolism

A. Interconversion of pyruvate and alanine by ALT

B. Alanine serves as a shuttle mechanism for carrying nitrogen to the liver

C. Other glucogenic amino acids that can be converted to pyruvate in the liver

IV. Malic Enzyme and Pyruvate Metabolism

A. Malic enzyme as a source of NADPH for lipogenesis

B. Importance of malic enzyme in liver cells

C. Conversion of malate to pyruvate and other metabolic pathways

V. Pyruvate Dehydrogenase (PDH) and Acetyl-CoA Formation

A. PDH catalyzes the oxidative decarboxylation of pyruvate

B. Fate of acetyl-CoA generated by PDH

C. Regulation of PDH activity

VI. Pyruvate Carboxylase and OAA Formation

A. Pyruvate carboxylase tags a CO2 onto pyruvate to form OAA

B. Role of pyruvate carboxylase in gluconeogenesis and TCA cycle replenishment

C. Regulation of pyruvate carboxylase activity

VII. Importance of Pyruvate Metabolism in Different Tissues

A. Consequences of PDH deficiency in tissues dependent on aerobic glucose oxidation

B. Role of pyruvate carboxylase

QUESTIONS

Qcard 1:

Question: What is the most important process for reoxidation of NADH in the cytoplasm of vertebrate cells under anaerobic conditions?

Answer: Conversion of pyruvate to lactate by lactate dehydrogenase (LDH).

Qcard 2:

Question: What is the pattern of LDH isozymes present in blood useful for distinguishing between?

Answer: Myocardial infarction and liver disease.

Qcard 3:

Question: What enzyme catalyzes the interconversion of pyruvate and alanine?

Answer: Alanine aminotransferase (ALT).

Qcard 4:

Question: What is the primary function of alanine in the body?

Answer: Incorporation into proteins and participation in transamination.

Qcard 5:

Question: What enzyme serves as an additional source of NADPH for lipogenesis?

Answer: Malic enzyme.

Qcard 6:

Question: What is the fate of acetyl-CoA generated by pyruvate dehydrogenase (PDH)?

Answer: Oxidation to CO2 and H2O via the TCA cycle or incorporation into other compounds.

Qcard 7:

Question: What regulates the activity of PDH?

Answer: Mitochondrial NADH/NAD+, ATP/ADP, GTP/GDP concentration ratios, acetyl-CoA levels, insulin, and pyruvate concentration.

Qcard 8:

Question: What is the consequence of PDH deficiency in tissues that depend on complete aerobic glucose oxidation for ATP generation?

Answer: Serious consequences, such as spino-cerebellar ataxia.

Qcard 9:

Question: What enzyme tags a CO2 onto pyruvate to form oxaloacetic acid?

Answer: Pyruvate carboxylase.

Qcard 10:

Question: What is the role of pyruvate carboxylase in maintaining appropriate TCA cycle intermediates in exercising muscle and other tissues?

Answer: Replenishing intermediates consumed in biosynthetic reactions and filling up the TCA cycle.

Mind Map: Metabolic Fates of Pyruvate

Central Idea: Metabolic pathways and enzymes involved in the conversion of pyruvate

Main Branches:

Anaerobic Metabolism
   * Conversion to lactate
   * Regeneration of NAD+
   * Importance in vertebrate cells
Amino Acid Metabolism
   * Conversion to alanine
   * Role in gluconeogenesis
   * Nitrogen transport to the liver
   * Other glucogenic amino acids
Malic Enzyme
   * Additional source of NADPH for lipogenesis
   * Transfer of reducing equivalents
   * Conversion to pyruvate or alanine
Pyruvate Dehydrogenase
   * Irreversible step in animal cells
   * Generation of acetyl-CoA
   * Oxidation or incorporation into other compounds
   * Regulation of PDH activity
Pyruvate Carboxylase
   * Conversion to oxaloacetic acid (OAA)
   * Role in gluconeogenesis
   * Replenishment of TCA cycle intermediates
   * Allosteric activation by acetyl-CoA

Sub-Branches:

Anaerobic Metabolism
  * Lactate Dehydrogenase (LDH)
    * Isozymes in blood
    * Differentiation of diseases
    * Inhibition by pyruvate
  * Alanine Aminotransferase (ALT)
    * Interconversion of pyruvate and alanine
    * Reversible reaction
    * Role in liver and muscle
Amino Acid Metabolism
  * Nitrogen transport to the liver
    * Formation of alanine
    * Gluconeogenesis in the liver
  * Other glucogenic amino acids
    * Tryptophan, glycine, serine, cysteine, threonine
Malic Enzyme
  * Decarboxylation reaction
  * Source of NADPH for lipogenesis
  * Transfer of reducing equivalents
  * Conversion to pyruvate or alanine
Pyruvate Dehydrogenase
  * Oxidative decarboxylation reaction
  * Generation of acetyl-CoA
  * Oxidation or incorporation into other compounds

Study Plan: Chapter 27: Metabolic Fates of Pyruvate

Day 1:

Read and understand the overall concept of metabolic fates of pyruvate.
Focus on the importance of anaerobic glycolysis and the conversion of pyruvate to lactate under anaerobic conditions.
Study the role of lactate dehydrogenase (LDH) in the reoxidation of NADH in the cytoplasm.
Take notes on the different LDH isozymes and their significance in diagnosing certain diseases.
Review the conversion of pyruvate to alanine through alanine aminotransferase (ALT) and its role in nitrogen transport.
Understand the shuttle mechanism of pyruvate and alanine for carrying nitrogen to the liver.

Day 2:

Review the role of malic enzyme in generating pyruvate in the cytoplasm and its importance in lipogenesis.
Understand the interconversion of malate and pyruvate and their various metabolic pathways.
Study the irreversible conversion of pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH).
Take note of the regulation of PDH activity and the factors that inhibit or stimulate its function.
Understand the fate of acetyl-CoA, including its oxidation in the TCA cycle and incorporation into other compounds.

Day 3:

Review the consequences of PDH deficiency in tissues dependent on aerobic glucose oxidation for ATP generation.
Focus on the role of pyruvate carboxylase in converting pyruvate to oxaloacetic acid (OAA) for gluconeogenesis.
Understand the energetics and cofactor requirements of the pyruvate carboxylase reaction.
Study the involvement of biotin and other factors in the pyruvate carboxylase reaction.
Take note of the high activity of pyruvate carboxylase in the liver and kidneys for gluconeogenesis.

Day 4:

Review the importance of acetyl-CoA as an allosteric activator of pyruvate carboxylase.
Understand the role of acetyl-CoA in replenishing the TCA cycle and facilitating citrate formation.
Study the significance of pyruvate carboxylase in maintaining appropriate TCA cycle intermediates in exercising muscle.