4 Stoichiometry of Glycolysis

Dr. Padma Ambalam

Stoichiometry of Glycolysis

Objectives

To understand the function of a central metabolic pathway that produces NADH and ATP during conversation of Glucose to pyruvate.
To examine the yield of total energy by chemical reaction.
To study the energy difference under aerobic and anaerobic condition.

Introduction

Glycolysis is the oldest of pathways evolved in allthe organisms.

It is divide into two phases and starts with glucose as principle substrate and produces 2-3 carbon substances as productGlyceraldehyde 3-phosphate (GAP).

In the second half, GAP is converted Pyruvate, which is further converted into acetyl CoA, ethanol or lactic acid depending presence or absence of oxygen.

Glycolysis is traditionally represented as starting with one molecule of glucose (i.e., C6H12O6) and ending with two molecules of pyruvate.

During this cycle, total a four ATP molecule are generated. However, a net gain is only two ATP moleculessince two ATP are used to “prime” the pathway.

The sugar-splitting step comes just prior to the conversion of NAD+ to NADH and also prior to the gain of four ATP (Figure 1).

The enzymes involved, in order, are

(1) Hexokinase (first ATP-priming step)

(2) Phosphoglucoseisomerase

(3) Phosphofructose kinase (second ATP-priming step)

(4) Aldolase (sugar-splitting step)

(5) Triphosphate isomerase

(6) Glyceraldehyde phosphate dehydrogenase (NAD+ → NADH step)

(7) Phosphoglycerase kinase (generation of two ATP per starting glucose)

(8) Phosphoglycerasemutase

(9) Enolase

(10) Pyruvate kinase (generation of two ATP per starting glucose)

Figure-1 A metabolic pathway of Glycolysis. (Adapted from http://ocw.osaka- u.ac.jp/engineering-jp/bioinformatics-jp/no-10-lecture-note.pdf)

Stoichiometry of Glycolysis Aerobic glycolysis Regeneration of NAD+:

1. Electron shuttles

Stoichiometry of steps 1-10:

In the cell NAD is present only in the limited, so regeneration of NAD+ is necessary. Moreover cytosolic NADH cannot enter mitochondria, so electron pair carried to mitochondrial electron transport chain via a shuttle (short linking pathway)

Net reaction:

NADHcyt + oxid e– carriermito → NAD+cyt + red. e– Carriermito

2 e–cyt → 2 e–mito

2. Malate-aspartate shuttle

It is amain shuttle in heart and liver cells
Electron pair eventually transferred to mitochondrial matrix NAD+, so ATP yield is 2.5/ electron pair

3. GOP-DHAP shuttle

It is a main shuttle in brain & skeletal muscle
Net reaction involves

NADHcyt + H+ + E-FAD→ NAD+cyt+ E-FADH2

yields 1.5 ATP per e– pair

Electrons with FAD, as FADH2, effect is the regenerate the NAD+, oxidized form, electrons passed to FAD, that has accomplished regeneration.

This is part of mitochondrial membrane so electrons transferred to coenzyme Q. This is where they join the electron transport chain. 1.5 ATP per pair of electrons. This is the entry point with complex II, skips the first complex, enters at lower level, so one of the ATP forming steps is skipped ,consequence is less ATP per pair of electrons. Depending on which shuttle is used, depending on cells, get more or less ATP produced.

ATP yield

(Steps 1-10) glucose + 2 NAD+ → 2 pyruvate + 2 NADH + 4H+ 2

Regeneration of NAD+: GOP shuttle + ox phos

2 H+ + 2 NADH + O2 → 2 NAD++ 2 H2O 3*

Glucose + O2 → 2 pyruvate + 2 H+ + 2 H2O 5

* 5 if malate-aspartate shuttle used

Table 1. Total ATP gain in Glycolysis

Step	Reaction	Consumption of ATP	Gain of ATP
1	Glucose→ Glucose 6-phosphate	1
3	Fructose6-phosphate → Fructose 1,6- diphosphate	1
7	1,3-diphosphoglycerate → 3- phosphoglycerate		1 × 2 = 2
10	Phosphoenolpyruvate → Pyruvate		1 × 2 = 2
		2	4
Net gain of ATP = 4 – 2 = 2

Anaerobic glycolysis:

Regeneration of NAD+: 1. reduction of pyruvate

Conditions limiting electron shuttles:
Mitochondria scarce (“fast” muscle) or absent (RBC)
Limited O2 supply (ischemia)
High demand for ATP causes glycolysis rate > shuttle rate
Electron pair is transferred to pyruvate:

As a result, glycolysis can occur without net oxidation: anaerobically Fermentation: any anaerobic process

Table 2.

	ATP yield
Glucose + 2 NAD+ → 2 pyruvate + 2 NADH + 4 H+ (Steps 1-10)	2
2 pyruvate +2 NADH + 2H+ → 2 lactate +2 NAD+ (Step 11)
Glucose → 2 lactate + 2 H+ (Steps 1-11)	2

In aerobic condition, take electrons away from NADH and transfer them to oxygen, to get ATP out, add these two reactions up, get glucose plus one oxygen, get to pyruvate, two protons, 7 if malate shuttle is used. Variation in ATP yield.

Anaerobic, no oxygen involved, so steps 1-10 are the same, anaerobically electrons transferred to pyruvate to make lactate, one glucose converted to two lactates and two protons, make lots of lactate, then also make lots of protons, where much of the acid comes from to dissolve enamel. Most yield of ATP under anaerobic conditi ons, but when there is no choice, then 2 is better than zero.

Table 3. Energy yield or use in every reaction.

Glycolytic reaction step
Glucose + ATP → Glucose 6- phosphate + ADP	-16.7	-33.4
Glucose 6 – phosphate ←→ Fructose 6 – phosphate	1.7	0 to 25
Fructose 6- phosphate + ATP → Fructose 1,6 – bisphosphate + ADP	-14.2	-22.2
Fructose 1,6 – bisphosphate ←→ Dihydroxyacetone phosphate + Gglyceraldehyde 3- phosphate	23.8	0 to -6
Dihydroxyacetone phosphate ←→ Glyceraldehyde 3- phosphate	7.5	0 to 4
Glyceraldehyde 3- phosphate + Pi + NAD+ ←→ 1,3- bisphosphoglycerate + NADH + H+	6.3	-2 to 2
1,3- Bisphosphoglycerate + ADP ←→ 3 – phosphoglycerate + ATP	-18.8	0 to 2
3 – phosphoglycerate ←→ 2 – phosphoglycerate	4.4	0 to 0.8
2 – phosphoglycerate ←→ phosphoenolpyruvate + H2O	7.5	0 to 3.3
Phosphoenolpyruvate + ADP → Pyruvate + ATP	-31.4	– 16.7

Overall reactions involved in Stoichiometry of Glycolysis

Each molecule of glucose yields 2 molecules of glyceraldehyde 3-phosphate, the overall 10 glycolytic reactions can be simplified as follows :

Glucose + 2 ATP + 2 Pi + 2 NAD+ + 2 H+ + 4 ADP → 2 pyruvate + 2 H+ + 4 ATP + 2 H2O

+ 2 NADH + 2 H+ + 2ADP

The net equation for glycolytic pathway, after removing the common terms would be

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

Following three steps are important and occursconcurrentlyduring glycolysis :

(a) Glucose is oxidized to pyruvate.

(b) Reduction of NAD+to NADH.

Energy yield of glycolysis

In the first step of glycolysis, hexokinase, a regulatory enzymes phosphorylates glucose with a utilization of one ATP molecule. This is step is important because (1) due to negative charge of Glucose 6-phosphate, it cannotpass through the membrane (2) Phosphorylation step would destabilize glucose and assisting its further metabolism.

The third step of glycolysis also involves phosphorylation. Fructose 6-phosphate is phosphorylated and converted into fructose 1, 6-biphosphate in the reaction catalysed by the enzyme phosphofructokinase (PFK). It is a key and second regulatory enzyme in glycolysis and sets the pace of the reaction pathway.

In the second stage of glycolytic pathway with the fructose 1,6-bisphosphate will split into glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). In the remaining steps of the pathway will have only three-carbon units.

The interconversion of GAP and DHAP is rapid and readily reversible and is catalysed by triose phosphate isomerase.

The sixth reaction is the payoff phase. GAP is converted to 1,3-biphosphoglycerate (1,3- BPG) in a reaction catalysed by Glyceraldehyde 3-phosphate dehydrogenase.

1,3- BPG is an acyl phosphate with a high phosphoryl-transfer potential. In the subsequent step of the glycolytic cycle, one of this phosphoryl groups is transferred.

This reaction was catalyzed by glyceraldehyde 3-phosphate dehydrogenase is really the sum of two processes: the oxidation of the aldehyde to a carboxylic acid by NAD+ and the joining of the carboxylic acid and orthophosphate to form the acyl -phosphate product.

In the tenth reaction of glycolysis, phosphoenolpyruvate (PEP) with high phosphorylation potential is converted to pyruvate and ATP is formed from ADP. This reaction is catalysed by Pyruvate kinase is also regulatory enzyme.

The stoichiometry of glycolysis is shown in table 4.

Table 4. Stoichiometry of whole Glycolysis.

Reaction

Enzyme

ΔG(kJ/mol) (in physiological conditions)

Thermodyna

mic equilibrium

constant

1. Glucose + ATP → Glucose 6- phosphate + ADP + H+

Hexokinase

-33.5

250

2. Glucose 6 – phosphate ←→ Fructose 6 – phosphate

Phosphogluco seisomerase

-2.5

0.45

3. Fructose 6- phosphate + ATP → Fructose 1,6 – bisphosphate + ADP + H+

Phosphofructo kinase

-22.2

242

4. Fructose 1,6 – bisphosphate ←→ Dihydroxyacetone phosphate + Glyceraldehyde 3- phosphate

Aldolase

-1.3

9.5 × 10-5

5.Dihydroxyacetone phosphate ←→ Glyceraldehyde 3- phosphate

Triose phosphate isomerase

+2.5

0.052

6. Glyceraldehyde 3- phosphate + Pi + NAD+ ←→ 1,3- bisphosphoglycerate + NADH + H+

Glyceraldehyd e 3-phosphate dehydrogenase

-1.7

0.089

7. 1,3- Bisphosphoglycerate + ADP ←→ 3 – phosphoglycerate + ATP

Phosphoglycer ate kinase

+1.3

57109

8. 3 – phosphoglycerate ←→ 2 –

phosphoglycerate

Phosphoglycer atemutase

+0.8

0.18

9. 2 – phosphoglycerte ←→ Phosphoenolpyruvate + H2O

Enolase

-3.3

0.49

10. Phosphoenolpyruvate + ADP → Pyruvate + ATP

Pyruvate kinase

– 16.4

10304

(Adapted from http://www.lce.hut.fi/teaching/S-114.2500/s2006/Glycellw.pdf)

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References

Pigman WW, Horton D (editors) : The Carbohydrates. Chemistry and Biochemistry. Academic Press, Inc., New York. 1972.
Caputto R, Barra HS, Cumar FA : Carbohydrate metabolism. Ann. Rev. Biochem. 36 : 211-246, 1967.

Web site

Books

Harper’sIllustrated Biochemistry by Robert K.. Murray, Daryl K. Granner, Peter A. Mayes, 26th Edition (2003)
Lehninger’S Principle of Biochemistry David L. Nelson and Michael M. Cox,5th Edition (2008)
Instant Notes: Biochemistry, 2nd Edition, B.D. Hames & N. M. Hooper (2005)