Glycolysis 1:Glycolysis consists of two stages, an ATP

investment stage, and an ATP earnings stage

Bioc 460 Spring 2008 - Lecture 25 (Miesfeld)

Lactate build-up can limit exercise

Metabolism of glucose by yeast under anaerobic conditions leads to the production of ethanol and CO2

• Glycolysis is an ancient pathway that cleaves glucose (C6H12O6) into two molecules of pyruvate (C3H3O3). Under aerobic conditions, the pyruvate is completely oxidized by the citrate cycle to generate CO2, whereas, under anaerobic (lacking O2) conditions, it is either converted to lactate, or to ethanol + CO2 (fermentation).

• The glycolytic pathway consists of ten enzymatic steps organized into two stages. In Stage 1, two ATP are invested to “prime the pump,” and in Stage 2, four ATP are produced to give a net ATP yield of two moles of ATP per mole of glucose.

• Three glycolytic enzymes catalyze highly exergonic reactions (G<<0) which drive metabolic flux through the pathway; these enzymes are regulated by the energy charge in the cell (ATP requirements). The three enzymes are hexokinase, phosphofructokinase 1, and pyruvate kinase.

• Glycolysis generates metabolic intermediates for a large number of other pathways, including amino acid synthesis, pentose phosphate pathway, and triacylglycerol synthesis.

Key Concepts in Glycolysis

The Four Metabolic Pathway Questions

1. What does glycolysis accomplish for the cell?

– Generates a small amount of ATP which is critical under anaerobic conditions.

– Generates pyruvate, a precursor to acetyl CoA, lactate, and ethanol (in yeast).

2. What is the overall net reaction of glycolysis?

Glucose + 2NAD+ + 2ADP + 2 Pi →2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O

ΔGº’ = -35.5 kJ/mol

The Four Metabolic Pathway Questions

3. What are the key regulated enzymes in glycolysis?

Hexokinase, Phosphofructokinase 1, Pyruvate kinase

4. What are examples of glycolysis in real life?

Glycolysis is the sole source of ATP under anaerobic conditions which can occur in animal muscle tissue during intense exercise. Fermentation also relies on glycolysis which is a process that is used to make alcoholic beverages when yeast cells are provided glucose without oxygen.

Where does glycolysis fit into the metabolic map?

Glycolysis is a central pathway that takes glucose generated by carbohydrate metabolism and converts it to pyruvate. Under aerobic conditions, the pyruvate is oxidized in the citrate cycle which generates reducing power for redox reactions in the electron transport system that result in ATP production by oxidative phosphorylation.

Glycolysis takes place entirely in the cytosol, whereas, pyruvate oxidation occurs in the mitochondrial matrix where ATP is generated. Oxygen is not required for glycolysis in the cytosol (anaerobic) but it is necessary for aerobic respiration in the mitochondrial matrix where the O2 serves as the terminal electron acceptor.

The complete oxidation of glucose to CO2 and H2O is highly favorable and releases a large amount of energy that can be harnessed for ATP synthesis

Glucose (C6H12O6) + 6O2 → 6CO2 + 6H2O

ΔGº’ = -2,840 kJ/molΔG = -2,937 kJ/mol

ΔGº’ for ATP synthesis = -30.5 kJ/mol

ΔG for ATP synthesis = ~-50 kJ/mol

Theoretical maximum yield = ~60 ATP/glucose

Actual yield = 32 ATP/glucose

Why are only 32 ATP generated out of a possible ~60 ATP?

Pyruvate can also be converted anaerobically to ethanol and CO2 by fermentation in some micoroorganisms, or converted to lactate

For every mole of glucose entering glycolysis, two moles of glyceraldehyde-3-P (GAP) are metabolized to pyruvate, generating in the process a net 2 ATP and 2 NADH.The NADH is a source of reducing power for the cell.

Overview of the Glycolytic Pathway

The two stages of glycolysis

The ATP investment stage generates the high energy intermediate glyceraldehyde-3-P (GAP) which is then oxidized to produce NADH and 1,3-bisphosphoglycerate. The next four reactions lead to the production of FOUR total ATP because each glucose molecule results in the production of TWO pyruvate. The net yield of ATP in glycolysis is therefore TWO ATP.

• Investment of 2 ATP

• Production of 2 Glyceraldehyde-3-P (GAP)

• The two highly regulated steps are hexokinase and phosphofructokinase 1 (both respond directly or indirectly to energy charge).

Stage 1

Each molecule of GAP

• Reducing power is captured in the form of NADH; this is a critical step.

• Phosphoglycerate kinase and pyruvate kinase catalyze a substrate level phosphorylation reaction yielding 4 ATP (2 net ATP).

• The two pyruvate molecules are further metabolized.

Stage 2

The six carbons and six oxygens present in glucose are stoichiometrically conserved by glycolysis in the two molecules of pyruvate that are produced. Hydrogen atoms in glucose are lost as H2O molecules and in the reduction of NAD+.

No loss of carbons or oxygen in glycolysis

Chemical features of the glycolytic reactions

• Ten enzymatic reactions– primarily bond rearrangements – phosphoryl transfer reactions– isomerizations– an aldol cleavage– an oxidation– a dehydration

• Ideally, you should know the names of all ten enzymes and the reactants and products. The names describe the metabolite structures, draw them if you like, or visualize them in your head.

• At the very minimum, you need to know which steps the ATP hydrolysis and synthesis takes place, the net reaction of glycolysis, and the three key enzymes the control glycolytic flux.

Free energy changes for the ten glycolytic reactions

Gº’ = -35.5 kJ/mol G = -72.4 kJ/mol

Reaction 1: Phosphorylation of glucose by hexokinase or glucokinase

Hexokinase is found in all cells.

A related enzyme with same enzymatic activity, glucokinase, is present primarily in liver and pancreatic cells.

Hexokinase binds glucose through an induced fit mechanism that excludes H2O from the enzyme active site

and brings the phosphoryl group of ATP into close proximity with the C-6 carbon of glucose

Why does it make sense that hexokinase is feedback inhibited by glucose-6-P when energy charge in the cell is high?

Hexokinase is feedback inhibited by glucose-6-P which binds to a regulatory site in the amino terminus of the enzyme

Reaction 2: Isomerization of glucose-6-P to fructose-6-P by phosphoglucose isomerase

Phosphoglucose isomerase (phosphohexose isomerase) interconverts an aldose (glucose-6-P) and a ketose (fructose-6-P) through a complex reaction mechanism that involves opening and closing of the ring structure.

Reaction 3: Phosphorylation of fructose-6-P to fructose-1,6-BP by phosphofructokinase 1

Reaction 3 is the second ATP investment reaction in glycolysis and involves the coupling of an ATP phosphoryl transfer reaction catalyzed by the enzyme phosphofructokinase 1 (PFK-1). This is a key regulated step in the glycolytic pathway because the activity of PFK-1 is controlled by numerous allosteric effectors (positive and negative).

Reaction 4: Cleavage of fructose-1,6-BP by aldolase to generate glyceraldehyde-3-P

and dihydroxyacetone-P

The splitting of fructose-1,6-BP into the triose phosphates glyceraldehyde-3-P and dihydroxyacetone-P is the reaction that puts the lysis in glycolysis (lysis means splitting).

Reaction 5: Isomerization of dihydroxyacetone-P to glyceraldehyde-3-P by triose phosphate isomerase

Glyceraldehyde-3-P, rather than dihydroxyacetone-P, is the substrate for reaction 6 in the glycolytic pathway, making this isomerization necessary.

The original TIM barrel structure

STAGE 2: ATP EARNINGS

Three key features of the very important stage 2 reactions:

1. Two substrate level phosphorylation reactions catalyzed by the enzymes phosphoglycerate kinase and pyruvate kinase generate a total of 4 ATP/glucose (net yield of 2ATP) in stage 2 of glycolysis.

2. An oxidation reaction catalyzed by glyceraldehyde-3-P dehydrogenase generates 2 NADH molecules that can be shuttled into the mitochondria to produce more ATP by oxidative phosphorylation.

3. Reaction 10 is an irreversible reaction that must be bypassed in gluconeogenesis by two separate enzymatic reactions catalyzed by pyruvate carboxylase and phosphoenolpyruvate carboxykinase

Reaction 6: Oxidation and phosphorylation of glyceraldehyde-3-P by glyceraldehyde-3-P

dehydrogenase to form 1,3-bisphosphoglycerate

The glyceraldehyde-3-P dehydrogenase reaction is a critical step in glycolysis because it uses the energy released from oxidation of glyceradehyde-3-P to drive a phosphoryl group transfer reaction using inorganic phosphate (Pi) to produce 1,3-bisphosphoglycerate.

1,3-bisphosphoglycerate has a change in standard free energy of hydrolysis that is higher than ATP hydrolysis

This difference in free energies is harnessed by the enzyme phosphoglycerate kinase in reaction 7 to drive the synthesis of ATP by a mechanism called substrate level phosphorylation.

Reaction 7: Generation of ATP by phosphoglycerate kinase in the conversion of

1,3-bisphosphoglycerate to 3-phosphoglycerate

Phosphoglycerate kinase catalyzes the payback reaction in glycolysis because it replaces the 2 ATP that were used in stage 1 to prime the glycolytic pathway.

Remember, this occurs twice for every glucose that entered glycolysis. This is an example of a substrate level [ADP] phosphorylation reaction, i.e., ATP synthesis that is not the result of aerobic respiration or photophosphorylation.

The molecular structure of phosphoglycerate kinase is similar to hexokinase in that it has two lobes (jaws) that each bind one of the substrates (ADP-Mg2+ or 1,3-bisphosphoglycerate) leading to a large conformational change in the enzyme that brings the substrates close together and excludes H2O from the active site.

Reactions 6 and 7 are coupled reactions!

Rxn 6 Glyceraldehyde-3-P + Pi + NAD+ → 1,3-bisphosphoglycerate + NADH + H+

ΔGº’ = +6.3 kJ/mol ΔG = -1.3 kJ/mol

Rxn 7 1,3-bisphosphoglycerate + ADP → 3-phosphoglycerate + ATP

ΔGº’ = -18.9 kJ/mol ΔG = +0.1 kJ/mol

Coupled Reactions (add Gº’ values)Glyceraldehyde-3-P + Pi + ADP + NAD+ → 3-phosphoglycerate + ATP + NADH + H+

ΔGº’ = -12.6 kJ/mol ΔG = -1.2 kJ/mol

Actual change in free energy (G) for each of these two reactions is very close to zero, and therefore both reactions are in fact reversible inside the cell. This is important for controlling flux through glycolysis and gluconeogenesis.

Reaction 8: Phosphoryl shift by phosphoglycerate mutase to convert 3-phosphyglycerate

to 2-phosphoglycerate

The purpose of reaction 8 is to generate a compound, 2-phosphoglycerate, that can be converted to phosphoenolpyruvate in the next reaction, in preparation for a second substrate level phosphorylation to generate ATP.

The mechanism of this highly reversible reaction requires a phosphoryl transfer from a phosphorylated histidine

residue (His-P) located in the enzyme active site

The metabolic intermediate 2,3-BPG can diffuse out of active site before it is converted to 2-phosphoglycerate.

Remember that 2,3-BPG is important in the regulation of oxygen binding by hemoglobin.

Reaction 9: Dehydration of 2-phosphoglycerate by enolase to form phosphoenolpyruvate (PEP)

The standard free energy for this reaction is relatively small (ΔGº’ = +1.7 kJ/mol) but it traps the phosphate group in an unstable enol form, resulting in a dramatic increase in the phosphoryl transfer potential of the triose sugar.

Standard free energy change for phosphate hydrolysis in 2-phosphoglycerate is ΔGº’ = -16 kJ/mol, whereas the standard freen energy change for phosphate hydrolysis of phosphoenolpyruvate it is an incredible ΔGº’ = -62 kJ/mol !

Reaction 10: Generation of ATP by pyruvate kinase when phosphoenolpyruvate is converted to pyruvate

The second of two substrate level phosphorylation reactions in glycolysis that couples energy released from phosphate hydrolysis (ΔGº’ = -62 kJ/mol) to that of ATP synthesis (ΔGº’ = +30.5 kJ/mol). Unlike phosphoenolpyruvate, pyruvate is a stable compound in cells that is utilized by many other metabolic pathways.

See if you can name all glycolytic enzymes and metabolites based on the chemical structures