Fundamentals

Glycolysis is employed by all tissues for the breakdown of glucose hence yielding ATP as an energy source and intermediates for other pathways. In cells with mitochondria and adequate O2 supply, Pyruvate is the end product of glycolysis. Pyruvate undergoes oxidative decarboxylation by pyruvate dehydrogenase enzyme to acetyl CoA, which enters the TCA cycle to yield ATP. In cells lacking mitochondria like RBCs or in hypoxia like AMI, aerobic glycolysis cannot take place. So, these cells use anaerobic glycolysis to reduce pyruvate to lactate while NADH converts to NAD.

Steps in glycolysis:

1) First, glucose needs to enter the cell. It does so by two mechanisms as follows:

a) Na Independent Facilitated Diffusion: In facilitated diffusion, glucose movement follows a concentration gradient, i.e., from high to low glucose concentration. It is mediated by 14 glucose transporters called GLUTs 1 to 14, which are tissue specific. GLUT 1 is in RBCs and BB barrier; GLUT 2 in the liver, kidney, and beta cells of the pancreas; GLUT 3 in neurons; GLUT 4 in adipose tissue and skeletal muscle (their number is increased by Insulin) and GLUT 5 is a Fructose transporter in testes and small intestine.

b) Na Monosaccharide Cotransporter System: This is an energy-requiring process as it transports glucose against a concentration gradient, i.e., from low to high glucose concentrations. The movement is coupled to the cotransport of Na hence also called SGLUT (Sodium GLUT). SGLUT is present in the renal tubules, choroid plexus, and intestine epithelial cells.

Once glucose is in the cell, it needs to be trapped by forming Glucose 6 Phosphate by either the enzyme Hexokinase or Glucokinase.

a) Hexokinase: Present in most tissues. It is inhibited by the enzyme product i.e. glucose 6 phosphate. Hexokinase has a low Km (high affinity) and low Vmax for glucose. This allows cells to use glucose even when the blood glucose levels are low. Hexokinase serves as a glucose sensor in the hypothalamus playing a key role in the adrenergic response to hypoglycemia.

b) Glucokinase: Present in liver parenchymal cells and beta cells of the pancreas. In beta cells of the pancreas, it serves as a glucose sensor, determining the threshold for insulin secretion. In the liver it facilitates glucose phosphorylation during hyperglycemia (post meal fed state). It has a higher Km ( low affinity) and high Vmax for glucose. Hence, glucokinase functions only when the hepatocyte glucose concentration is elevated e.g. post meal. Activity is inhibited indirectly by fructose 6 phosphate (2^nd step in glycolysis) and not by glucose 6 phosphate and is indirectly stimulated by glucose. Mutations that decrease the activity of glucokinase can cause maturity onset diabetes of the young or MODY.

Role of GKRP (glucokinase regulatory protein in the liver): In the presence of fructose 6 phosphate, glucokinase is translocated to the nucleus, where it binds to GKRP and becomes inactive. When glucose levels rise, glucokinase is released from the GKRP and becomes active. Fructose 1 phosphate activates glucokinase by inhibiting the formation of glucokinase - GKRP complex.

2) Glucose 6 phosphate to Fructose 6 phosphate by phosphoglucose isomerase.

3) Fructose 6 Phosphate to Fructose 1,6 Biphosphate by PFK 1 is rate limiting and the most important step in the regulation of glycolysis. PFK 1 or phosphofructokinase 1 is inhibited by elevated levels of ATP, citrate, and activated by AMP and fructose 2,6 biphosphate.

Fructose 2,6 biphosphate is formed by the enzyme PFK 2 by a reversible reaction. Fructose 2,6 biphosphate stimulates glycolysis while at the same time inhibiting gluconeogenesis (by inhibiting fructose 1, 6 bisphosphatase, a gluconeogenesis enzyme). During the well-fed state, when insulin is high/ post meal and glucagon is low (cAMP is low, inactive protein kinase A causing dephosphorylation i.e. activation of PFK 2), fructose 2, 6 biphosphate levels increase stimulating glycolysis. While during fasting/starvation, when insulin is low, and glucagon is high, fructose 2,6 biphosphate levels in the liver are low, thus inhibiting glycolysis and activating gluconeogenesis.

4) Fructose 2,6 biphosphate is cleaved by the enzyme aldolase to glyceraldehyde 3 phosphate (G3P) and DHAP (dihydroxyacetone phosphate).

5) Triose phosphate Isomerase converts G3P to DHAP.

6) G3P is converted to 1,3 BPG by the enzyme Glyceraldehyde 3 phosphate dehydrogenase, also forming NADH.

Arsenic Poisoning: Inhibits Pyruvate dehydrogenase by binding to Lipoic Acid. Pentavalent arsenic (arsenate) prevents the formation of NADH and ATP in glycolysis by competing with inorganic phosphate as a substrate for G3PDehydrogenase. Arsenic also inhibits the ATP Synthase enzyme.

2,3 BPG: In RBCs, 1,3 BPG is converted to 2,3 BPG by the enzyme BPG mutase, facilitating increased oxygen delivery to tissues.

7) 1,3 BPG to 3 phosphoglycerate by phosphoglycerate kinase. ATP is produced.

8) 3 phosphoglycerate is converted to 2 phosphoglycerate by phosphoglycerate mutase.

9) 2 phosphoglycerate to PEP (phosphoenolpyruvate) by enolase

10) PEP to pyruvate by pyruvate kinase. ATP is formed. In the liver, pyruvate kinase is activated by fructose 1,6 biphosphate (feed–forward regulation). When blood glucose levels are low, elevated glucagon increases cAMP, activating protein kinase A and causing phosphorylation and inactivation of pyruvate kinase.

The metabolic steps of glycolysis and TCA cycle. The steps involved in glycolysis and the TCA cycle are demarcated separately. ATP/GTP utilization or synthesis is shown in green, while NAD + /NADH and FAD/FADH 2 are shown in red. Also indicated are the numbers of NADH, FADH 2, and ATP/GTP generated when one molecule of glucose is consumed following glycolysis, TCA cycle, and oxidative phosphorylation.

Pyruvate Kinase Deficiency: Mature RBCs lack mitochondria and depend totally on glycolysis for energy/ ATP. Decreased ATP causes damage to the RBC membrane and altered shape, ultimately leading to phagocytosis by splenic macrophages and hemolytic anemia. RBCs compensate by increasing 2,3 BPG. In pyruvate kinase deficiency, the enzyme pyruvate kinase is mutated and shows altered kinetics like abnormal response to activator fructose 1,6 biphosphate; abnormal Km or Vmax; abnormal folding, reduced enzyme amounts etc.

Pyruvate to Lactate: Lactate is the end product of anaerobic glycolysis by enzyme lactate dehydrogenase seen in lens, cornea, renal medulla, testes, leukocytes and RBCs.

In exercising skeletal muscle, NADH production exceeds the oxidative capacity of the respiratory chain. This results in increased NADH/NAD ratio, favoring the reduction of pyruvate to lactate by Lactate dehydrogenase or LDH (which catalyzes a bidirectional reaction). Lactic acid in the muscle may cause cramps.

When the ratio of NADH/NAD is low, like in heart muscle and liver, there is oxidation of lactate from blood to pyruvate by LDH. In the liver, pyruvate is either converted to glucose by gluconeogenesis or oxidized in the TCA cycle.

Measurement of lactic acid levels is an indicator of “oxygen debt” which predicts mortality and morbidity in many clinical conditions.