Glycolysis and Gluconeogenesis

Hemolytic Anemia

In the process of its maturation, the red blood cell ( RBC ) eliminates many of its subcellular structures, including the cell nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus to maximize storage capacity for hemoglobin ( Hb ). Because of the lack of mitochondria for oxidative metabolism, the RBC is dependent on glycolysis for its energy [in the form of adenosine triphosphate (ATP)], and its metabolism needs to be intact for the function of the cell membrane as well as for the structure and functions of Hb. Most of the energy for the RBC is generated by glycolysis (up to 90%) where two ATPs are generated from the metabolism of a glucose molecule. Other pathways in the RBC are the pentose phosphate pathway (PPP) (or hexose monophosphate shunt) that metabolizes 5%–10% of glucose available and generates nicotinamide adenine dinucleotide phosphate (NADPH) , the methemoglobin (mHb) reductase pathway, and the Rapoport–Luebering Shunt .

Virtually any deficiency of an enzyme in the glycolytic pathway would seriously imperil the lifetime (normally 120 days) of an RBC owing to the fact that the RBC depends upon this pathway almost entirely for energy. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH, 37 kDa) catalyzes the sixth reaction in the glycolytic pathway, the conversion of glyceraldehyde-3-phosphate to 1,3- bis phosphoglycerate (1,3-BPG), and the generation of nicotinamide adenine dinucleotide (NADH)+H ⁺ from NAD ⁺ . In addition to catalyzing the glycolytic step, G3PDH may function as an activator of transcription in that it has been shown to be part of the OCA-S [complex is a gene(s)-encoding histones] coactivator complex (in addition to lactate dehydrogenase, also a part of the complex). The G3PDH has additional functions: it can move between the cytosol and the nucleus; it is able to initiate apoptosis and is also able to bind to a nuclear ubiquitin ligase directing a target protein for degradation.

The absence of G3PDH means that the RBC dies before its natural lifetime. Impairment of the function of the RBC causes many symptoms, such as shortness of breath, dizziness, headache, cold extremities, pale skin, chest pain, fatigue, arrhythmias, enlarged heart, and heart failure. Also, because RBCs are dying early, Hb is released into the bloodstream (whereas it is usually collected by the spleen at the end of a normal lifetime) and it is metabolized to bilirubin (yellow) that can represent jaundice. Excessive bilirubin, especially when combined with high levels of cholesterol, can generate stones in the gallbladder, spleen enlargement, and abdominal pain. The treatment of hemolytic anemia may require blood transfusions. Severe hemolytic anemia can be fatal.

Methemoglobin Reductase Pathway

mHb contains oxidized iron (Fe ³⁺ ), a form that is unable to bind oxygen. It is only a trace in blood representing about 1%. The NADPH (reduced form) mHb reductase pathway maintains the oxygen-binding form of Hb that contains iron in the Fe ²⁺ ferrous form ( Fig. 8.1 ).

Rapoport–Luebering Shunt

2,3- Bis phosphoglycerate ( 2,3-BPG ) is an intermediate between 1,3-BPG and 3-phosphoglycerate. This intermediate, 2,3-BPG , is an allosteric effector of Hb that regulates the affinity of Hb for oxygen and facilitates the release of oxygen to the tissues (e.g., lungs). The 2,3-BPG binds to the beta subunit of the T (taut) state of Hb, deoxyhemoglobin, the less active form. The pocket in which 2,3-BPG binds measures 11 Angstroms for deoxyhemoglobin (T state), whereas the same pocket in oxyhemoglobin (R state, relaxed) measures 5 Å; 2,3-BPG itself measures about 9 Å, so it can fit into the T state pocket but not in the R state pocket of the Hb beta subunit. The complex enhances the ability of oxyhemoglobin to release oxygen to the needy tissues (e.g., lungs). The concentration in the RBC of 2,3-BPG is determined by the activities of bis phosphoglycerate mutase and 2,3-BPG phosphatase ( Fig. 8.2 ).

A lowered pH in the RBC is inhibitory to 2,3-BPG mutase and stimulatory to 2,3-BPG phosphatase. Consequently, under this condition, there would be more 1,3-BPG available to form 3-phosphoglycerate through the phosphoglycerate kinase reaction and generate ATP.

The Pentose Phosphate Pathway

The PPP is outlined in Fig. 6.26. It is summarized in its connections to other major pathways: glycolysis, glycogen metabolism, and the tricarboxylic acid (TCA) cycle in Fig. 8.3 .

The various functions of the PPP can provide NADPH from NAD ⁺ and ribose-5-phosphate for the ultimate synthesis of nucleic acids. NADH is needed to reduce GSSG (two glutathione molecules joined by a disulfide bridge; the oxidized form of glutathione) to GSH (glutathione), particularly in cells, such as the RBCs that are subject to oxidative stresses and the production of H ₂ O ₂ and free peroxy-radicals. The PPP is, in a sense, elastic in that it can adapt to the needs of a particular cell at a point in time when the metabolism of a cell is requiring reducing equivalents in the form of NADPH, or needing to divide that requires DNA and RNA and the production by the PPP of ribose-5-phosphate, or needing to synthesize lipid from the same three-carbon intermediates of glycolysis, or needing energy in the form of ATP. Thus the four modes of PPP function are shown diagrammatically in Fig. 8.4 .

Glycolysis, the Emden–Meyerhof Pathway

Glycolysis is a metabolic process in which glucose (or other sugars that can funnel into the pathway) is converted to a series of intermediates leading to the formation of pyruvic acid . In muscle, for example, as oxygen becomes used up through activity, pyruvic acid, so formed, can be converted to lactic acid under anaerobic conditions by the lactate dehydrogenase reaction. In glycolysis, two molecules of pyruvic acid are formed per glucose molecule. The reverse direction of the pathway, leading to the formation of glucose, is gluconeogenesis. Gluconeogenesis is not strictly a reversal of glycolysis because there are some different unique enzymatic steps involved at the points of irreversible reactions . The pathways of glycolysis and gluconeogenesis are shown in Fig. 8.5A and B

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In glycolysis, two ATPs are used and four ATPs are generated providing a net of two ATPs in the conversion of one molecule of glucose to two molecules of pyruvate. One ATP is used in the hexokinase/glucokinase reaction converting glucose to fructose-6-phosphate. Hexokinase is primarily used in muscle and glucokinase (an isozyme of hexokinase, hexokinase IV ) is used preferentially in liver. Glucokinase has a larger Km (10 mM) for glucose than does hexokinase (0.2 mM) allowing glucokinase to handle larger amounts of glucose. Glucokinase in liver shuttles between the nucleus and the cytoplasm. When the cellular level of glucose is low or when fructose-6-phosphate level is high, glucokinase is transported into the nucleus. Conversely, when the level of glucose is elevated in the cell, glucokinase is transported to the cytoplasm to initiate glycolysis.