9.2. Glycolysis occurs mainly in 3 stages:
- Stage 1 is the investment stage because in these steps two molecules of ATP are consumed for each molecule of glucose. Glucose converted into unstable form that can be cleaved into 3 carbon units.
- Stage 2 is preparatory stage in which fructose 6 phosphate is cleaved into 2, 3 carbon units of glyceraldehyde 3 phosphate.
- Stage 3 is the harvesting stage in which 4 molecules of ATP and 2 molecules of NADH are gained from each initial moles of glucose. Glyceraldehyde 3 phosphate is oxidized to pyruvate.
9.2.1. The Hexokinase Reaction:
The first reaction of glycolysis is the ATP-dependent phosphorylation of glucose to form glucose 6-phosphate (G6P), and is catalyzed by tissue-specific isoenzymes known as hexokinases.
The phosphorylation achieves two goals:
- Phosphorylation converts non-ionic glucose into an anion that is trapped in the cell, since cells lack transport systems for phosphorylated sugars.
- Biologically inert glucose becomes activated into a labile form capable of being further metabolized.
Hexokinase binds with glucose and cosubstrate ATP. After the binding it adopts a closed conformation, in which substrate and cosubstrate are buried within the enzyme and water is excluded from the active site. Hexokinase has two lobes that form its active site cleft for glucose. On glucose binding, lobes moves towards each other. The bound glucose of carbon 6, which accepts the phosphorylate group from ATP. Galactose is different from glucose only in the position of the OH group. For the metabolism of galactose, cells require a separate galactokinase that mean enzyme specificity is maintained. Binding of glucose causes conformational changes in enzymes. It has a low km for glucose. Hexokinase envelopes its substrates to prevent ATP hydrolysis.
Glucokinase is found in hepatocytes and pancreatic β-cells. Glucokinase has high Km for glucose means that this enzyme is saturated only at very high concentrations of substrate.
The Km for hexokinase is significantly lower (0.1mM) than that of glucokinase (10mM). Low Km for glucose indicates high affinity. Hexokinase is expressed in non-hepatic tissues. This low Km allows rapid and efficient trap of blood glucose by all cell except liver. After meals, when blood glucose levels are high, liver glucokinase is significantly active. Thus liver trap excess glucose from hepatic portal vain and protect the cell from hyperglycaemic situation.
The normal circulating concentration of glucose is 5mM and the Km of glucokinase is 10mM. The higher Km (low affinity) of glucokinase for glucose allows glucose to leave the hepatocyes because glucokinase has low affinity for glucose lower than normal circulating concentration of glucose. Hence liver delivers glucose to the blood and maintain the blood glucose level.
During starvation when blood glucose falls to very low levels, glucose is not uptaken by liver and kidney because their glucokinases has low affinity (high Km) for glucose. At the same time, tissues such as the brain, which is critically dependent on glucose, continue to uptake blood glucose using their low Km hexokinases.
If the enzyme is given xylose instead of glucose, one water molecule can squeeze into the active site along with the sugar. This water assumes the place of the C6 hydroxymethyl group of glucose, and it will be activated by hexokinase to react with ATP, which will result in ATP hydrolysis. But the phosphate is not transfer to xylose however ATP is hydrolysed.
9.2.2. Phosphoglucose isomerase:
Phosphohexose isomerase provides an excellent example of acid-base catalysis which involves the reversible protonation and deprotonation of the substrate. It converts an aldolase into a ketose. Isomerization of glucose -6-phosphate (an aldose) into a fructose -6-phosphate (Ketose). Both aldose and ketose are present primarily in the cyclic form, so the enzymes must first open the six numbered ring of glucose-6-phosphate, catalyses the isomerization and then promote the formation of the five member ring of fructose-6-phosphate.
Reaction mechanism of phosphoglucose isomerase. The active site catalytic residues (BH+ and B’) are thought to be Lys and a His-Glu dyad respectively.
The enzyme phosphofructokinase uses another ATP molecule to transfer a phosphate group to fructose 6-phosphate on C1 to form fructose 1, 6-bisphosphate. This allosteric enzyme plays a central role in control of Glycolysis. This is the rate determining reaction of glycolysis.
9.2.4. Aldolase :
The fructose ring opens and breaks into two different sugar phosphates which are isomers to each other. These two sugars are dihydroxyacetone phosphate (ketose) and glyceraldehyde phosphate(aldose). There are two types of aldolases. Class one aldolases occur in animals and plants. Class two aldolases occur in fungi, algae and some bacteria. They do not form a (a sub-class of imines) schiff base with the substrate.
This enzyme interconverts DHAP and GAP, which are ketose- aldose isomers. GAP is a substrate for the next step in glycolysis so all of the DHAP is eventually depleted. So, two molecule of GAP are formed from each molecule of glucose. It shows rest half part of glycolysis from GAP to pyruvate formation.
9.2.6. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH):
It forms 1, 3 bis phosphoglycerate (3BPG). GAPDH has an active site cis sulfhydral group. GADPH inactivates by alkylation with stoichiometric amounts of iodoacetate. GADPH quantitatively transfer 3H from C1 of GAP to NAD+.
9.2.7. Phosphoglycerate Kinase:
This step is first ATP generation reaction. It transfers a phosphate group from 1, 3-bisphosphoglycerate to ADP to form ATP and 3-phosphoglycerate (3PG). It is a substrate- level- phosphorylation reaction requires ADP. Thus when cell has plenty of ATP, this reaction does not occur as the substrate ADP is not available for the reaction.
9.2.8. Phosphoglycerate mutase:
It converts 3 PG into 2 PG (2 phosphoglycerate). A mutase catalyzes the transfer of a functional group from one position to another on a molecule.
It converts 2PG into PEP (phosphoenolepyruvate). Its cofactor is Mg+2. Firstly it binds with Mg+2 then with second metal divalent ion. Catalytic mechanism of enolase occur in one of three ways:
- By generating a carbonation at C-3. Firstly the OH group at C-3 leaves for it.
- By generating carbonation at C-2, so C-2 proton can leave first.
- Paul Boyers isotope exchange study shows that the C-2 proton of 2PG exchanges with solvent 12 times faster than the rate of PEP formation. However, the C-3 oxygen exchanges with solvent at a rate roughly equivalent with the overall reaction rate.
9.2.10. Pyruvate kinase:
It is second ATP generation reaction. It transfers a phosphate group from PEP to ADP to form ATP and pyruvate. The standard free energy of hydrolysis of 2PG is only -17.6 kj/mole, which is insufficient to drive ATP synthesis (-30.5 kj/mole for ATP synthesis from ADP and Pi). The standard free energy of hydrolysis of PEP is -61.9 kj/mole. That is sufficient for ATP synthesis. So PEP is a “high energy” compound.
Energy-rich functional groups in substrates of glycolysis
- The enolphosphate in PEP
- The carboxyphosphate in 1,3-bisphosphoglycerate
- The thioester in the active site of glyceraldehyde-3-dehydrogenase
9.3. Fates of Pyruvate
9.3.1. Anaerobic fate of pyruvate (fermentation)
Reduction of pyruvate can occur in two ways homolactic and alcoholic fermentation which occurs in muscles and yeast. Respectively depends on the reduction pathway of NAD+. The NAD+ formed during glycolysis should be reduced in NADH and GAPDH. In the presence of oxygen, the reducing equivalents of NADH passed into mitochondria for re-oxidation. Under anaerobic conditions, the NAD+ reduced in above mentioned form.
9.3.2. Homolactic Fermentation
This is a feature of muscles, occur particularly during vigorous muscle contraction. In rapidly contracting muscle cells, the demand for energy is high. After the O2 supply is depleted, lactic acid fermentation provides sufficient NAD+ to allow glycolysis (with its low level of ATP production) to continue for a short time.
9.4. Recycling of NADH During Anaerobic Glycolysis
Lactate dehydrogenase (LDH) catalyzes the oxidation of NADH by pyruvate to yield NAD+ and lactate. The NADH produced during the conversion of glyceraldehyde-3-phosphate to glycerate-1,3-bisphosphate is converted into NAD+ when pyruvate is converted to lactate. This process allows the cell to continue producing ATP under anaerobic conditions as long as glucose is available.
LDH works with absolute stereo specificity. The pro-R (A side) hydrogen at C4 of NADH is stereospecifically transferred to the pyruvate at C2 to form L- (or S-) lactate. This regenerates NAD+ for participation in the GAPDH reaction. The hydride transfer to pyruvate is from the same face of the nicotinamide ring as that to acetaldehyde in the alcohol dehydrogenase reaction but from the opposite face of the nicotinamide ring as that to GAP in the GADPH reaction.
Mammals have two different types of LDH subunits, the M (M type predominant in tissues that are subjected to anaerobic conditions such as skeletal muscles and liver) and H type (predominant in aerobic tissue like heart muscles), which together form five tetrameric isozymes: M4, M3H, M2H2, MH3, H4. H4 LDH has low km for pyruvate and allosterically inhibited by high level of this metabolites. Whereas M4 LDH has high Km for pyruvate, is not inhibited by it. The other isozymes have intermediate properties that vary with the ratio of their two types of subunits. So the H type LDH is better for oxidation of lactate to pyruvate and M type LDH is better to catalyse the reverse reaction.
Glucose +2ADP+2Pi= 2-lactate +2ADP +2H2O+2H+
9.5. The Crabtree Effect
The glucose represses aerobic metabolism, is the Crabtree effect. This is a feature of S. cerevisiae (yeast).
S. cerevisiae is a facultative anaerobe:
It can produce ATP both in the presence and the absence of O2.
When the glucose or fructose levels rise, pyruvate is diverted away from the citric acid cycle (the first phase of aerobic energy generation) into ethanol synthesis by conversion to acetaldehyde and CO2 by pyruvate decarboxylase.
When glucose levels are high, yeast cells shift into the “make, accumulate, consume” ethanol pathway.
- Glucose is converted into ethanol molecules to regenerate NAD+.
- Ethanol is then released into the environment where it kills competing microbes.
- Excess ethanol is stored inside the cell.
- Once glucose is depleted, glucose repression ends.
- Now cell synthesized ADH-2 enzyme that converts ethanol back into acetaldehyde.
- Acetaldehyde is subsequently converted into acetyl-CoA.
- Acetyl-CoA enter into the citric acid cycle.
Note that although the “make, accumulate, consume” strategy is expensive yeast cells manage to kill off the competition and retrieve a waste product that they then use as an energy source.
9.6. Energy Yield of Aerobic Versus Anaerobic Glycolysis
In both aerobic and anaerobic glycolysis, each mole of glucose generates two molecules of ATP, two molecules of NADH and two molecules of Pyruvate. The net energy yield in anaerobic glycolysis is two molecules of ATP per molecule of glucose, as the NADH is recycled to NAD+ by reducing pyruvate to lactate. When oxygen is available and cytosolic NADH can be oxidized via a shuttle system from mitochondria, pyruvate can also enter into the mitochondria and be completely oxidized to CO2 via PDH and TCA cycle. One NADH gives 2.5 ATP if is oxidized by the malate aspartate shuttle. One NADH gives 1.5 ATP if is oxidized by the glycerol 3-Phosphate shuttle.
Thus, the two NADH molecules produced during glycolysis can lead to 3 to 5 molecules of ATP being produced, depending on which shuttle system is used to transfer the reducing equivalents. Because each pyruvate produced can give rise to 12.5 molecules of ATP, altogether 30 to 32 molecules of ATP.
Tissue dependent on anaerobic glycolysis
Many tissues including red and white blood cells, the kidney medulla, the tissues of the eyes, and skeletal muscles, relay on anaerobic glycolysis for at least a portion of their ATP requirements.
Some of the lactic acid generated by anaerobic glycolysis in skin is secreted in sweat, where it acts as an antibacterial agent. Many large tumours use anaerobic glycolysis for ATP production and lack capillaries in their core.
9.7. Cori Cycle
In the liver the pyruvate is used to synthesis glucose (gluconeogenesis) which is returned to the blood. The cycling of lactate and glucose between peripheral tissues and liver is called the cori cycle.
Glucose is an indispensable metabolite
- The brain requires at least ~50% of its calories in the form of glucose
- Red blood cells exclusively dependent upon glucose
- Glucose is a precursor of other sugars needed in the biosynthesis of nucleotides, glycoproteins and glycolipids
- Glucose is needed to replenish NADPH, which supplies reducing power for biosynthesis and detoxification
Note: These considerations make the need for gluconeogenesis quite clear—we just can’t afford to leave the blood glucose level up to the vagaries of dietary supply.