Glycolysis is the sequence of reactions that metabolize one molecule of glucose into two molecules of pyruvate with the production of two molecules of ATP
Anaerobic no O 2 needed aerobic O 2 needed
In 1897 Hans and Eduard Buchner discovered that yeast extracts could rapidly ferment sucrose into alcohol. Startling discovery in its day because it was widely held that fermentation was a process that occurs within cells. This led to efforts to understand the mechanism of this extracellular fermentation. Studies in muscle extracts also revealed that lactic acid fermentation was very similar to alcohol fermentation in yeast. The complete glycolytic pathway was finally elucidated by 1940 through the pioneering contributions of Embden, Meyerhof, Neuberg, Parnas, Warburg and G&C Cori. Glycolysis is also known as the Embden-Meyerhof pathway.
Glucose is an important fuel for most organisms. In mammals glucose is used by the brain under nonstarvation conditions and is the only fuel used by red blood cells. There are many different monosaccharides but glucose predominates as the primary fuel.
Why is glucose such a prominent fuel in all life forms? 1. Glucose may have been available for primitive biochemical systems because it can form under prebiotic conditions. 2. Glucose is the most stable hexose. 3. Glucose has a low tendency to nonenzymatically glycosylate proteins.
Glucose is generated from dietary carbohydrates: Pancreatic a-amylase cleaves the 1,4 bonds of starch: the products are maltose and maltotriose. Maltase cleaves maltose into two glucose molecules and a-glucosidase cleaves maltotriose into three glucose molecules. Maltase and a-glucosidase are located on the surface of the small intestine these monosaccharides are now transported into the blood stream.
Glycolysis converts one molecule of glucose into two molecules of pyruvate with the generation of two molecules of ATP. Glycolysis can be thought of as occurring in two stages: 1. Stage 1 traps glucose in the cell and modifies it so that it can be cleaved into a pair of phosphorylated 3- carbon compounds. 2. Stage 2 oxidizes the 3-carbon compounds to pyruvate while generating two molecules of ATP.
Upon entering the cell through a specific transport protein, glucose is phosphorylated at the expense of ATP to form glucose 6-phosphate. Hexokinase, which requires Mg 2+ or Mn 2+ as a cofactor, catalyzes the reaction. Hexokinase, like most kinases, employs substrate-binding induced fit to minimize hydrolysis of ATP.
Hexokinase places glucose into a cleft and then the cleft closes trapping glucose and excluding H 2 O
The conversion of glucose 6-phosphate to fructose 6-phosphate is catalyzed by phosphoglucose isomerase. The reaction is readily reversible.
A second phosphorylation step occurs after isomerization catalyzed by phosphofructokinase: Bisphosphate means separate phosphate groups
The carbohydrate is trapped in the fructose form by the addition of a second phosphate to form fructose 1,-6 bisphosphate. This irreversible reaction is catalyzed by the allosteric enzyme phosphofructokinase (PFK).
The 6-carbon F-1,6-BP is cleaved by aldolase into G3P and DHAP
G3P is on a direct pathway of glycolysis whereas DHAP is not and needs to be converted into G3P by an isomerase: At equilibrium 96% of the triose phosphate is DHAP but utilization of G3P by glycolysis forces the reaction to generate G3P
Glu165 and His95 play key roles in the reaction Glu removes the H+ from C1 (1) and His donates a H+ to the O bound to C2 (2) then Glu donates the H+ to C2 (3)
TPI traps the enediol intermediate with the adjacent loop of the enzyme preventing loss of the intermediate which would lead to the production of a toxic methyl glyoxal molecule
The enediol intermediate may potentially decompose into the very reactive methyl glyoxal. Methyl glyoxal release is prevented by structural changes in the enzyme. Triose phosphate isomerase is a catalytically perfect enzyme because its rate of catalysis is near the diffusion limit.
Phase two of glycolysis begins when a compound with high phosphoryl transfer potential, 1, 3-bisphosphoglycerate, is generated by the oxidation of GAP in a reaction catalyzed by glyceraldehyde 3- phosphate dehydrogenase.
The formation of glyceraldehyde 1,3-bisphosphate can be thought of as occurring in two steps: the highly exergonic oxidation of carbon 1 in GAP to an acid, and the highly endergonic formation of glyceraldehyde 1, 3- bisphosphate from the acid. These two reaction are linked by the formation of an energy-rich thioester in the active site of glyceraldehyde 3-phosphate dehydrogenase.
Catalytic mechanism of glyceraldehyde 3-phosphate dehydrogenase Cys reacts with the aldehyde group of the substrate Oxidation takes place with the transfer of H+ to NAD forming a thioester intermediate NADH is replaced by NAD+ Orthophosphate attacks the thioester forming 1,3-BPG
Cysteine reacts with the aldehyde group of G3P
Oxidation results in the transfer of a hydride ion to NAD with the formation of the thioester
NADH is replaced by NAD+
NAD+ facilitates the attack of orthophosphate on the thioester
1, 3-Bisphosphoglycerate has a greater phosphoryl-transfer potential than ATP, and thus can be used to power the synthesis of ATP from ADP and P i in a reaction catalyzed by phosphoglycerate kinase.
3-Phosphoglycerate is converted into 2-phosphoglycerate by phosphoglycerate mutase. A dehydration reaction, catalyzed by enolase, results in the production of phosphoenolpyruvate (PEP). Phosphoenolpyruvate is a high phosphoryl-transfer compound because the presence of the phosphate traps the compound in the unstable enol tautomer. ADP is phosphorylated at the expense of PEP, generating ATP and pyruvate, in a reaction catalyzed by pyruvate kinase.
Additional ATP is generated with the formation of pyruvate dehydration
The reaction catalyzed by the mutase involves a phosphorylated enzyme intermediate and catalytic amounts of 2, 3-bisphosphoglycerate. The net reaction is:
Additional ATP is generated with the formation of pyruvate dehydration
The phosphoryl group present in the enol form generated by the action of enolase is unstable; once transferred to ADP by pyruvate kinase it rapidly undergoes conversion to the ketone pyruvate. The driving force for this reaction is large because of this conversion.
Glucose+2Pi+2ADP+2NAD == 2pyruvate+2ATP+2NADH+2H+
The conversion of glucose into pyruvate generates ATP, but for ATP synthesis to continue, NADH must be reoxidized to NAD +. This vital coenzyme is derived from the vitamin niacin (B 3 ). NAD + can be regenerated by further oxidation of pyruvate to CO 2, or by the formation of ethanol or lactate from pyruvate.
Ethanol is formed from pyruvate in yeast and other microorganisms Lactate is formed in a variery of microorganisms and also in cells of higher organisms when 0 2 is limiting Much more energy is extracted by conversion of pyruvate to acetyl CoA via the citric acid cycle
The regeneration of NAD + by processing pyruvate to ethanol is called alcoholic fermentation.
Alcohol dehydrogenase requires zinc ion as a cofactor.
Pyruvate to ethanol regenerates NAD+
The regeneration of NAD+ by the alcohol and lactate fermentation keeps a redox balance
In lactic acid fermentation, pyruvate is reduced to lactate to regenerate NAD +.
The three dehydrogenases that we have considered are very different structurally but all three possess a conserved NAD+ binding domain often called the Rossmann fold
Some organisms cannot live in the presence of O 2
Fructose, from table sugar or high-fructose corn syrup, and galactose from milk sugar are converted into glycolytic intermediates. In the liver, fructose is metabolized by the fructose 1-phosphate pathway. In other tissues, such as adipose tissue, fructose is directly phosphorylated by hexokinase.
Other sugars 4 enzymatic steps 1 enzymatic step
Fructose in the liver enters the glycolytic pathway via the fructose 1-phosphate pathway
Fructose is a commonly used sweetener. Excess consumption of fructose has been linked to fatty liver, insulin insensitivity, obesity, and type 2 diabetes. In the liver, fructose metabolism bypasses the key regulatory enzyme phosphofructokinase. The excess pyruvate is converted into acetyl CoA and then into fatty acids.
Galactose is converted into glucose 6-phosphate by the galactose-glucose conversion pathway, which begins with the phosphorylation of galactose by galactokinase.
Entry of galactose into glycolysis utilizes a previously modified galactose molecule
Glucose 1-phosphate is isomerized to glucose 6- phosphate by phosphoglucomutase And is now on the glycolytic pathway
Lactose intolerance or hypolactasia occurs because most adults lack lactase, the enzyme that degrades lactose. Northern Europeans have a mutation that prevents the decline of lactase activity after weaning. In lactase-deficient individuals, gut bacteria metabolize lactose, generating CH 4 and H 2, and disrupt water balance in the intestine.
Classic galactosemia results if galactose 1-phosphate uridyl transferase activity is deficient. Symptoms include failure to thrive, jaundice, and liver enlargement that can lead to cirrhosis. Cataract formation may also occur.
A deficency of galactose 1-phosphate uridyl transferase can lead to toxic levels of galactose; in the eye lens galactose is converted into galactitol causing the production of a cataract
The Glycolytic pathway is tightly controlled The rate of conversion of glucose to pyruvate is tightly regulated. In glycolysis the reactions catalyzed by hexokinase, phosphofructokinase and pyruvate kinase are essentially irreversible making them targets for regulation. These enzymes become more or less active in response to the binding of allosteric effectors (milliseconds) or covalent modifications (seconds). In addition the levels of these enzymes are regulated by the transcription of their genes (minutes).
Hexokinase Phosphofructokinase Pyruvate kinase Are irreversible but regulated
Phosphofructokinase is the key regulator of glycolysis in mammals. The enzyme is allosterically inhibited by ATP and allosterically stimulated by AMP. When ATP needs are great, adenylate kinase generates ATP from 2 ADP. AMP then becomes the signal for the low-energy state.
Phosphofructokinase is regulated by the ratio of ATP/AMP A tetramer of 4 identical subunits
Hexokinase is allosterically inhibited by glucose 6-phosphate. Pyruvate kinase is inhibited by the allosteric signals ATP and alanine, and stimulated by fructose 1, 6-bisphosphate, the product of the phosphofructokinase reaction. In muscle, glycolysis is regulated to meet the energy needs of contraction.
The regulation of glycolysis in the liver is more complex than in muscle tissue The liver has more diverse biochemical functions than muscle. The liver maintains blood-glucose levels and stores glucose as glycogen when glucose levels are plentiful. Phosphofructokinase regulation with respect to ATP is similar to muscle, however low ph is not a metabolic signal. In the liver phosphofructokinase is inhibited by citrate, an early intermediate in the citric acid cycle.
The key regulators of phosphofructokinase in liver are citrate, which reports on the status of the citric acid cycle, and fructose 2, 6-bisphosphate. Citrate inhibits phosphofructokinase whereas fructose 2,6-bisphosphate is a powerful activator.
The regulation of glycolysis in the liver is more complex than in muscle tissue A high level of citrate indicates that biosynthetic intermediates are abundant so there is no need to degrade additional glucose for this purpose. Citrate inhibits phosphofructokinase by enhancing the inhibitory effect of ATP.
Glycolysis in the liver responds to changes in blood glucose through the signaling molecule; fructose 2,6-bisphosphate. F-2,6-BP is a potent activator of phosphofructokinase. In the liver the concentration of fructose 6-phosphate rises when blood glucose is high, the abundance of fructose 6-phosphate accelerates the synthesis of F-2,6-BP. F-2,6-BP increases the affinity of phosphofructokinase for F6P and diminishes the inhibitory effect of ATP. Thus glycolysis is accelerated when glucose is abundant, a process known as feed-forward stimulation.
Feed-forward stimulation in response to high blood glucose levels by the liver
F-2,6-BP stimulates phosphofructokinase even at low substrate concentrations (A). ATP inhibitory effects are attenuated in the presence of F-2,6-BP (B).
Hexokinase is an allosteric enzyme in the liver as it is in muscle. The enzyme primarily responsible for phosphorylating glucose in the liver is glucokinase. Glucokinase is active only after a meal, when blood-glucose levels are high. Pyruvate kinase in the liver is regulated allosterically as it is in muscle. However, liver pyruvate kinase is also regulated by covalent modification. Low blood glucose leads to the phosphorylation and inhibition of liver pyruvate kinase.
The liver isoforms of pyruvate kinase can be modified by phosphorylation which reduce its activity and limit the livers utilization of glucose saving it for muscle and brain to consume.
Five glucose transporters, termed GLUT1-5, facilitate the movement of glucose across the cell membrane.
GLUT s consist of a single chain of 500 amino acid isoforms:
Rapidly growing tumors obtain ATP by metabolizing glucose to lactate even in the presence of oxygen, a process termed aerobic glycolysis or the Warburg effect. When patients are infused with a non-metabolizable analog of glucose, tumors are readily visualized by tomography.
The transcription factor hypoxia-inducible transcription factor 1 (HIF-1) facilitates aerobic glycolysis. Exercise training also stimulates HIF-1, which enhances the ability to generate ATP anaerobically and stimulates new blood vessel growth.
Glucose can be synthesized from non-carbohydrate precursors The gluconeogenesis pathway converts pyruvate into glucose. The major non-carbohydrate precursors are lactate, amino acids and glycerol. Lactate formed in muscle tissue and can be readily converted into pyruvate by the enzyme lactate dehydrogenase. Amino acids are derived from proteins in the diet. Glycerol is converted into dihydroxyacetone phosphate then enters the pathway via conversion to glyceraldehyde 3-phosphate
Pathway of Glycolysis The three irreversible steps of glycolysis prevents the simple reversal of glycolysis to convert pyruvate to glucose
Pathway of Gluconeogenesis
Hexokinase Phosphofructokinase
Pyruvate kinase
Conversion of glycerol to dihydroxyacetone phosphate
Pyruvate is converted into oxaloacetate by a mitochondrial enzyme: pyruvate carboxylase
Pyruvate carboxylase uses the vitamin biotin as a cofactor. The formation of oxaloacetate by pyruvate carboxylase occurs in three stages. 1. The biotin carboxylase domain catalyzes the formation carboxyphosphate. 2. The carboxylase then transfers the CO 2 to the biotin carboxyl carrier protein (BCCP). 3. The BCCP carries the activated CO 2 to the pyruvate carboxylase domain, where the CO 2 is transferred to pyruvate to form oxalacetate. Acetyl CoA is a required cofactor for carboxylation of biotin.
PC
Biotin-binding domain of pyruvate carboxylase; biotin is covalently attached through an e-nitrogen atom of lysine
Biotin contains an activated CO 2 molecule
pyruvate carboxylase Malate dehydrogenase
Oxaloacetate is converted into phosphoenolpyruvate by an ER bound enzyme: phosphoenolpyruvate carboxykinase
Glucose 6-phosphate is converted to glucose in the ER by glucose 6-phosphatase This process occurs primarily in the liver:
The stoichiometry of gluconeogenesis: 2pyruvate+4ATP+2GTP+2NADH+6H2O= glucose+4adp+2gdp+6pi+2nad+2h + DG=-11 kcal/mole
Energy charge determines whether glycolysis or gluconeogenesis will be most active The most important regulatory site is the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate When energy is needed the concentration of AMP will be relatively high. AMP stimulates phosphofructokinase and inhibits fructose 1,6-bisphosphatase, glycolysis is favored. Conversely high levels of ATP and citrate indicate there are abundant biosynthetic intermediates. ATP and citrate inhibit phosphofructokinase and activate fructose 1,6-bisphosphatase, gluconeogenesis is favored.
Low energy High energy
Glycolysis and gluconeogenesis are also reciprocally regulated at the interconversion of phosphoenolpyruvate and pyruvate in the liver The glycolytic enzyme pyruvate kinase is inhibited by allosteric effectors ATP and alanine, signaling energy charge is high and building blocks are abundant. Conversely pyruvate carboxylase, which catalyzes the first step of gluconeogenesis from pyruvate is inhibited by ADP. Similarly ADP inhibits phosphoenolpyruvate carboxykinase. Gluconeogenesis is favored when a cell has ample ATP and biosynthetic precursors.
The balance between glycolysis and gluconeogenesis in the liver is sensitive to blood-glucose concentrations The signaling molecule fructose 2,6-bisphosphate strongly stimulates phosphofructokinase(pfk) and inhibits fructose 1,6- bisphosphatase When blood-glucose is low fructose 2,6-bisphosphate looses a phosphate to form fructose 6-phosphate which can not allosterically influence PFK. The level of fructose 2,6-bisphosphate is critical in determining whether glycolysis or gluconeogenesis will occur. Two enzymes regulate the concentration of this molecule: one phosphorylates fructose 6-phosphate and the other dephosphorylates fructose 2,6- bisphosphate.
Domain structure of the bifunctional enzyme phosphofructokinase 2
The activities of PFK2 and FBP2 are reciprocally controlled by the phosphorylation of a single serine residue At low glucose levels a rise in the hormone glucagon triggers a camp signaling cascade leading to the phosphorylation of the bifunctional enzyme by protein kinase A. This modification activates FBPase2 and inactivates PFK2 leading to a drop in the levels of F-2,6-BP resulting in gluconeogenesis. When blood glucose levels are high, insulin is secreted by the pancreas initiating a signaling pathway that activates a protein phosphatase removing the phosphoryl group from the bifunctional enzyme thus PFK2 is activated and FBP2 inhibited and glycolysis begins as the levels of F-2,6-BP rise.
Promoter structure of the phosphoenolpyruvate carboxykinase gene IRE insulin response element (repression) GRE glucocorticoid response element TRE thyroid hormone response element CREI and CREII camp response elements (activation)
The Cori cycle
Lactate is converted to pyruvate by lactate dehydrogenase Isozymic forms of lactate dehydrogenase in different tissues catalyze the inter-converson of pyruvate to lactate. The two primary isoforms are H (predominates in the heart) and M (predominates in skeletal muscle and the liver). These subunits associate to form 5 types of tetramers the H 4 has higher affinities for substrates than the M 4. H 4 oxidizes lactate to pyruvate so the heart is always aerobic. M 4 is optimized to convert pyruvate into lactate to allow glycolysis to proceed under anaerobic conditions.