Krebs Cycle with Molecular Models

Krebs Cycle with Molecular Models

The Krebs cycle (alias, the citric acid cycle, alias the tricarboxylic acid cycle), when reduced to its most fundamental purpose, generates reducing power in the form of NADH and FADH2. It does this by “dissecting off” hydrogens from two carbon fragments remaining after glucose goes through glycolysis and subsequent decarboxylation of pyryvate yielding acetyl coenzyme A. The acetyl group is fed into the cycle by attachment to oxaloacetate, yielding citrate.

Here is an overview of the molecules involved in the Krebs cycle

Krebs_Cycle

What follows are molecular models of the sequential molecules involved in the Krebs Cycle:

00_AcetylCoA_oxaloacetate_PB120039
Oxaloacetate Acetyl CoA transfers its acetyl group to the number two carbonyl carbon via the methyl end forming citrate
01_citrate_PB120030
Citrate Note that it has a tertiary alcohol which is not oxidizable.
02_Isocitrate_PB120024
Isocitrate The hydroxyl has been shifted so that it is now a secondary alcohol, and can be oxidized.
03_alpha_ketoglutarate_PB120025
Alpha ketoglutarate When Isocitrate is oxidized, leading to the reduction of NAD+, it also is decarboxylated
04_Succinyl_CoA_PB120033
Succinyl CoA In a reaction similar to the formation of acetyl CoA, ketoglutarate is oxidized, decarboxylated and a CoA attached. (Note that the coenzyme A moiety is indicated by a turquoise group
05_Succinate_PB120034
Succinate The thioester bond in succinyl CoA is hydrolyzed forming fumarate, with generation of GTP linked to the process.
06_fumarate_PB120029
Fumarate Succinate is dehydrogenated, forming trans fumarate with the concomitant reduction of FAD to FADH. (Why isn’t this molecule in the cis configuration? Anyone?)
07_Malate_PB120036
Malate Water is added to fumarate, leading to the formation of a secondary alcohol.
08_Oxaloacetate_PB120021
Oxaloacetate The alcohol is oxidized (similar to oxidation of isocitrate), reducing NAD+ to NADH, forming oxaloacetate.

Glycolysis/Fermentation with Molecular Models

Glycolysis/Fermentation with Molecular Models

“Glycolysis” strikes fear into many undergrad biology students because it presents them with an abstract series of reactions and molecules which are difficult to visualize and therefore incorporate into a coherant biochemical framework. This exercise has each student taking responsibility for a single molecule in the series, learning the following about it:

1) its structure
2) its precursor
3) the enzyme which created it
4) the enzyme which will act on it
5) the product of that action
6) the significance of the bond structure, particularly those involving phosphate. (Note whether phosphoester (low energy) or phosphoanhydride (high energy).)

They are then to describe these features to their fellow students in sequence. This strategy for teaching glycolysis has received many positive reviews from students who have used it.

See the bottom of the page for a key to the elements and directions for the construction of the models.

Here is the sequential listing of the molecules of glycolysis

01_glucose_P9280144

1) Glucose

Glucose is both an aldose and a hexose. It enters the cell by diffusion, and the action of hexokinase holds it there.

Hexokinase transfers a phosphate from ATP to the number six carbon on glucose. This not only initiates glycolysis, but traps glucose in the cell since the ionic phosphate group makes diffusion out of the cell impossible without assistance.

02_glucose-6-phosphate_P9280145

2) Glucose-6-phosphate (G-6-P)

Phosphoglucoisomerase moves the carbonyl from the number one to the number two carbon, changing the molecule from an aldose to a ketose. This will free up the number one carbon for the phosphoryllation of the next step.

03_fructose-6-phosphate_P9280146

3) Fructose-6-phosphate (F-6-P)

Phosphofructokinase-1 is a critical enzyme in several ways. It transfers a phosphate from ATP to the number one carbon, thus placing ionic “handles” on either end (a PO4 on both the number 1 and number 6 carbons), allowing for the “breaking” of the molecule in the next step. This enzyme is allosterically inhibited by elevated ATP concentration.

04_fructose-1-6-bis_phosphate_P9280147

4) Fructose-1,6-bisphosphate (F1,6bisP)

Aldolase splits fructose-1,6-bisphosphate into two pieces, dihydroxyacetonephosphate (DHAP, a ketone) and glyceraldehyde-3-phosphate of the next step.

05_action_of_aldolase_P928014806_DHAP_and_glyceraldehyde-3-phosphate_P9280150

5) Dihydroxyacetone phosphate (DHAP)

The image at the upper left shows fructose 1,6-phosphate at the bottom, splitting into dihydroxyacetone phosphate above on the left, and glyceraldehye-3-phosphate above on the right.

Triose phosphate isomerase moves the carbonyl from the number two carbon to the number one carbon, isomerizing DHAP to glyceraldehyde-3-phosphate. Thus, a single glucose generates two glyceraldehude-3-phosphates and all the following reactions are doubled.

The lower image shows dihydroxyacetone phosphate above on the left, and glyceraldehye-3-phosphate close up.

07_glyceraldhyde-3-phosphate_P9280152

6) Glyceraldehyde-3-phosphate (3-GAP)

Glyceraldehyde-3-phosphate dehydrogenase performs a complex reaction in which glyceraldehyde-3-phosphate is oxidized by the removal of the hydrogen from the aldehyde. This hydrogen is used to reduce NAD+. A phosphate is added to the number one carbon in place of the hydrogen. This produces a very high energy phosphoacid anhydride.

08_1-3_bis_phosphoglycerate_P9280153

7) 1,3-bisphosphoglycerate (1,3bisPGA)

Phosphoglycerokinase transfers the high energy phosphate from the phosphoanhydride bond on the number one carbon to an ADP (substrate level phosphoryllation).

Note that this is the first ATP to be generated, and two are created for every glucose molecule which entered the pathway.

09_3-phosphoglycerate_P9280154

8) 3-phosphoglycerate (3-PGA)

Phosphoglyceromutase moves the phosphate from the number three carbon to the number two carbon to prepare it for dehydration in the step after next.

10_2-phosphoglycerate_P9280155

9) 2-phosphoglycerate (2-PGA)

Enolase dehydrates 2-phosphoglycerate to form phosphoenolpyruvate.

thmb_11_phosphoenolpyrvate_P9280156

10) Phosphoenolpyruvate (PEP)

Pyruvate kinase transfers the high energy phosphate from PEP to ADP, yielding pyruvate and ATP.

Phosphoenolpyruvate is the most energetic molecule in all of the molecules in glucose catabolism, because of the strain in the enol and the phosphate being adjacent to an ethylene bond.

It can engage in substrate level phosphoryllation, donating its phosphate to an ADP yielding ATP.

12_pyruvic_acid_P9280157

11) Pyruvate

Pyruvate is the end product of glycolysis, and, in the presense of oxygen, will be dehydrogenated by pyruvate dehydrogenase to yield acetyl coenzyme A, the “crossroads” molecule of carbon metabolism.

12_lactate_PB121155

12) Lactic acid

Lactate dehydrogenase regenerates NAD+ (required for oxidation of glyceraldehyde-3-phosphate by (named for the opposite reaction) reducing pyruvate. This happens in muscle during intense exercise (ergo, muscle burn) and in milk during fermentation (butermilk and yogurt).

13_acetaldehyde_PB121156

13) Acetaldehyde

Pyruvate decarboxylase decarboxylates pyruvate in yeast to produce acetaldehyde. Thiamine is required for decarboxylation, and yeast synthesizes it in large quantities, making nutritional yeast an excellent source for this vitamin.

14_ethanol_CO2_PB121158

14) Carbon dioxide & Ethanol

Alcohol dehydrogenase oxidizes acetaldehyde and concomitantly oxidizes NADH to yield ethyl alcohol and NAD+, required for oxidation of glyceraldehude-3-phosphate thus allowing glycolysis to continue in the absense of oxygen or other hydrogen acceptor.

Once students have constructed the assigned molecule, and learned the enzymes and related molecules, the models are laid out in sequence on a big table, and students in succession gave the following information:

1) The name of the molecule they had constructed
2) Its characteristic features
3) How it differs from the previous molecule
5) The enzyme which produced it
6) How it will be changed into the next molecule and why
7) The name of the enzyme which performs this change and the meaning of the name of the enzyme.

Students were to discuss selected phosphorylated molecules (i.e. glucose 6 phosphate, 1,3 bisphosphoglycerate, and phosphoenolpyruvate), naming and discussing the bonds by which phosphate is attached, and the relative energy content in each type (phosphoester, phosphoanhydride, resons for PEP’s unusual energy). The products of hydrolysis of these bonds was demonstrated and discussed.

Following the discussion, students leave the room, the molecules are randomized, and students return and identify the molecules as a way of demonstrating what they have learned.

KEY TO CONSTRUCTION OF MODEL MOLECULES:

Here is the key for the identities of the elements in the models

For the construction, remember that in straight chain illustrations of the chemical structures:
The carbon back bone is vertical with vertical bonds from each carbon projecting away from the observer.

The horizontal bonds project towards the observer.

Review the lab protocol on the Krebs cycle molecules.

Videos on glycolysis from Films for the Humanities and Sciences series which review the process are:

VIDT QH 633 .C45 1992 pt.1 Cell and Energy (Series Title-Cellular Respiration)
VIDT QH 633 .C45 1992 pt.2 Glycolosis 1 (Series Title-Cellular Respiration)
VIDT QH 633 .C45 1992 pt.3 Glycolosis 2 (Series Title-Cellular Respiration