The second phase of glycolysis involves the extraction of energy in the form of 4 ATP per molecule of glucose, a net gain of 2 ATP molecules. The product of glycolysis, pyruvate, can then be further broken either aerobically ( to carbon dioxide and water through the TCA cycle) or anaerobically (to lactate or alcohol).
Catalyses: Glyceraldehyde-3-phosphate (G3P) +NAD+ + Pi à 1,3-Bisphosphoglycerate (1,3BPG) + NADH + H+
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis and gluconeogenesis by reversibly catalysing the oxidation and phosphorylation of G3P to the energy-rich intermediate 1,3BPG. NAD+ is a co-substrate for this reaction.
GAPDH displays diverse non-glycolytic functions as well, its role depending upon its subcellular location. For instance, the translocation of GAPDH to the nucleus acts as a signalling mechanism for programmed cell death, or apoptosis. The accumulation of GAPDH within the nucleus is involved in the induction of apoptosis, where GAPDH functions in the activation of transcription. The presence of GAPDH is associated with the synthesis of pro-apoptotic proteins like BAX, c-JUN and GAPDH itself.
GAPDH has been implicated in certain neurological diseases: GAPDH is able to bind to the gene products from neurodegenerative disorders such as Huntington’s disease, Alzheimer’s disease, Parkinson’s disease and Machado-Joseph disease through stretches encoded by their CAG repeats. Abnormal neuronal apoptosis is associated with these diseases. Propargylamines such as deprenyl increase neuronal survival by interfering with apoptosis signalling pathways via their binding to GAPDH, which decreases the synthesis of pro-apoptotic proteins.
Catalyses: 1,3-Bisphosphoglycerate (1,3BPG) + ADP à 3-Phosphoglycerate (3PG) + ATP
Phosphoglycerate kinase (PGK) is an enzyme that reversibly catalyses the formation of ATP to ADP, using one of the high-energy phosphate groups from 1,3BPG. The reaction forms two ATP molecules per glucose (one per 1,3BPG molecule), which compensates for the expenditure of 2 ATP in phase I of glycolysis. The ATP is made by substrate-level phosphorylation, where a phosphate group is transferred from 1,3BPG directly to ADP. This reaction is essential in most cells for the generation of ATP in aerobes, for fermentation in anaerobes and for carbon fixation in plants.
Deficiencies in PGK are associated with haemolytic anaemia, myopathy, central nervous system disorder and growth retardation.
Catalyses: 3-Phosphoglycerate (3PG) à 2-Phosphoglycerate (2PG)
Phosphoglycerate mutase (PGAM) catalyses the transfer of the phospho group from the C3 position to the C2 position, in preparation for the synthesis of ATP. PGAM enzymes from different sources exhibit different reaction mechanisms. For instance, some PGAM enzymes (vertebrates, fungi, certain bacteria) use 2,3-bisphophoglycerate as a cofactor to phosphorylate a serine residue to prime the reaction, whereas other PGAM enzymes (plants, certain invertebrates, algae, certain bacteria) carry out intramolecular phosphoryl group transfer via an active site residue without the need of a cofactor.
Deficiencies in PGAM can cause acute muscle dysfunction with exercise intolerance and muscle breakdown.
Catalyses: 2-Phosphoglycerate (2PG) à Phosphoenolpyruvate (PEP) + H2O
Enolase (phosphopyruvate hydratase) is an essential glycolytic enzyme that catalyses the reversible dehydration of 2-phosphoglycerate to the high-energy intermediate phosphoenolpyruvate. Enolase is strongly inhibited by fluoride ions, which forms a fluorophosphate complex with magnesium at the active site. In vertebrates, there are 3 different, tissue-specific isozymes, designated alpha, beta and gamma. Alpha is present in most tissues, beta is localised in muscle tissue, and gamma is found only in nervous tissue. The functional enzyme exists as homo- or hetero-dimers of the different isozymes. In immature organs and in adult liver, it is usually an alpha homodimer, in adult skeletal muscle, a beta homodimer, and in adult neurons, a gamma homodimer. In developing muscle, it is usually an alpha/beta heterodimer, and in the developing nervous system, an alpha/gamma heterodimer.
Neuron-specific enolase is released in a variety of neurological diseases, such as multiple sclerosis and after seizures or acute stroke. Several tumour cells have also been found positive for neuron-specific enolase. Beta-enolase deficiency is associated with glycogenosis type XIII defect.
Catalyses: Phosphoenolpyruvate (PEP) + ADP à Pyruvate + ATP
Pyruvate kinase (PK) catalyses the final step in glycolysis, the conversion of PEP to pyruvate with the concomitant transfer of the high-energy phosphate group from PEP to ADP, thereby generating ATP. PK requires both magnesium and potassium for activity. In vertebrates, there are four tissue-specific isozymes: L (liver), R (red cells), M1 (muscle, heart and brain), and M2 (early foetal tissue). In plants PK exists as cytoplasmic and plastid isozymes, while most bacteria and lower eukaryotes have one form, except in certain bacteria, such as Escherichia coli, that have two isozymes.
PK helps control the rate of glycolysis, along with phosphofructokinase and hexokinase. PK possesses allosteric sites for numerous effectors, yet the isozymes respond differently, in keeping with their different tissue distributions. The activity of L-type (liver) PK is increased by fructose-1,6-bisphosphate (F1,6BP) and lowered by ATP and alanine (gluconeogenic precursor), therefore when glucose levels are high, glycolysis is promoted, and when levels are low, gluconeogenesis is promoted. L-type PK is also hormonally regulated, being activated by insulin and inhibited by glucagon, which covalently modifies the PK enzyme. M1-type (muscle, brain) PK is inhibited by ATP, but F1,6BP and alanine have no effect, which correlates with the function of muscle and brain, as opposed to the liver.
The pyruvate produced by PK feeds into a number of different metabolic pathways. Under aerobic conditions, pyruvate can be transported to the mitochondria, where it enters the TCA cycle and is further broken down to produce considerably more ATP through oxidative phosphorylation. Alternatively, pyruvate can be anaerobically reduced to lactate in cells lacking mitochondria, or under hypoxic conditions, such as found in hard working muscle tissues. Other by-products of anaerobic breakdown of pyruvate include ethanol during fermentation by yeast