Step 1.  Acyl-CoA Dehydrogenase catalyzes oxidation of the fatty acid moiety of acyl-CoA, to produce a double bond between carbon atoms 2 and 3.

There are different Acyl-CoA Dehydrogenases for short (4-6 C), medium (6-10 C), long and very long (12-18 C) chain fatty acids. Very Long Chain Acyl-CoA Dehydrogenase is bound to the inner mitochondrial membrane. The others are soluble enzymes located in the mitochondrial matrix.

FAD is the prosthetic group that functions as electron acceptor for Acyl-CoA Dehydrogenase. 

A glutamate side-chain carboxyl extracts a proton from the a-carbon of the substrate, facilitating transfer of 2 e^- with H⁺ (a hydride) from the b position to FAD. The reduced FAD accepts a second H⁺, yielding FADH₂

The carbonyl oxygen of the thioester substrate is hydrogen bonded to the 2'-OH of the ribityl moiety of FAD, giving this part of FAD a role in positioning the substrate and increasing acidity of the substrate a-proton

The reactive glutamate and FAD are on opposite sides of the substrate at the active site. Thus the reaction is stereospecific, yielding a trans double bond in enoyl-CoA.

FADH₂ of Acyl CoA Dehydrogenase is reoxidized by transfer of 2 electrons to an Electron Transfer Flavoprotein (ETF), which in turn passes the electrons to coenzyme Q of the respiratory chain.

Step 2. Enoyl-CoA Hydratase catalyzes stereospecific hydration of the trans double bond produced in the 1st step of the pathway, yielding L-hydroxyacyl-Coenzyme A

Step 3. Hydroxyacyl-CoA Dehydrogenase catalyzes oxidation of the  hydroxyl in the b position (C3) to a ketone. NAD⁺ is the electron acceptor.

Step 4. b-Ketothiolase (b-Ketoacyl-CoA Thiolase) catalyzes thiolytic cleavage.

A cysteine S attacks the b-keto C. Acetyl-CoA is released, leaving the fatty acyl moiety in thioester linkage to the cysteine thiol. The thiol of HSCoA displaces the cysteine thiol, yielding fatty acyl-CoA (2 C shorter).

A membrane-bound trifunctional protein complex with two subunit types expresses the enzyme activities for steps 2-4 of the b-oxidation pathway for long chain fatty acids. Equivalent enzymes for shorter chain fatty acids are soluble proteins of the mitochondrial matrix.

Summary of one round of the b-oxidation pathway:

fatty acyl-CoA + FAD + NAD⁺ + HS-CoA → 
            fatty acyl-CoA (2 C shorter) + FADH₂ + NADH + H⁺ + acetyl-CoA

The b-oxidation pathway is cyclic. The product, 2 carbons shorter, is the input to another round of the pathway. If, as is usually the case, the fatty acid contains an even number of C atoms, in the final reaction cycle butyryl-CoA is converted to 2 copies of acetyl-CoA

ATP production:

• FADH₂ of Acyl CoA Dehydrogenase is reoxidized by transfer of 2 e^- via ETF to coenzyme Q of the respiratory chain. H⁺ ejection from the mitochondrial matrix that accompanies transfer of 2 e^- from CoQ to oxygen, leads via chemiosmotic coupling to production of approximately 1.5 ATP. (Approx. 4 H⁺ enter the mitochondrial matrix per ATP synthesized.)
• NADH is reoxidized by transfer of 2 e^- to the respiratory chain complex I. Transfer of 2 e^- from complex I to oxygen yields approximately 2.5 ATP.
• Acetyl-CoA can enter Krebs cycle, where the acetate is oxidized to CO₂, yielding additional NADH, FADH₂, and ATP. 
• Fatty acid oxidation is a major source of cellular ATP

b-Oxidation of very long chain fatty acids also occurs within peroxisomes

FAD is electron acceptor for peroxisomal Acyl-CoA Oxidase, which catalyzes the first oxidative step of the pathway. The resulting FADH₂ is reoxidized in the peroxisome producing hydrogen peroxide FADH₂ + O₂ à FAD + H₂O₂

The peroxisomal enzyme Catalase degrades H₂O₂ by the reaction:
2 H₂O₂ → 2 H₂O + O₂
These reactions produce no ATP

Once fatty acids are reduced in length within the peroxisomes they may shift to the mitochondria to be catabolized all the way to CO₂. Carnitine is also involved in transfer of fatty acids into and out of peroxisomes

Glycolysis Pathway

The reactions of Glycolysis take place in the cytosol of cells.

Glucose enters the Glycolysis pathway by conversion to glucose-6-phosphate. Initially, there is energy input corresponding to cleavage of two ~P bonds of ATP. 

1. Hexokinase catalyzes:  glucose + ATP → glucose-6-phosphate + ADP

ATP binds to the enzyme as a complex with Mg⁺⁺.

The reaction catalyzed by Hexokinase is highly spontaneous 

2. Phosphoglucose Isomerase catalyzes: 

glucose-6-phosphate (aldose) → fructose-6-phosphate (ketose)

The Phosphoglucose Isomerase mechanism involves acid/base catalysis, with ring opening, isomerization via an enediolate intermediate, and then ring closure .

3. Phosphofructokinase catalyzes: 

fructose-6-phosphate + ATP  → fructose-1,6-bisphosphate + ADP

The Phosphofructokinase reaction is the rate-limiting step of Glycolysis. The enzyme is highly regulated. 

4. Aldolase catalyzes: 

fructose-1,6-bisphosphate   → dihydroxyacetone phosphate + glyceraldehyde-3-phosphate

The Aldolase reaction is an aldol cleavage, the reverse of an aldol condensation.

5. Triose Phosphate Isomerase (TIM) catalyzes

dihydroxyacetone phosphate (ketose) → glyceraldehyde-3-phosphate (aldose)

Glycolysis continues from glyceraldehydes-3-phosphate

The equilibrium constant (K_eq) for the TIM reaction favors dihydroxyacetone phosphate, but removal of glyceraldehyde-3-phosphate by a subsequent spontaneous reaction allows throughput. 

6. Glyceraldehyde-3-phosphate Dehydrogenase catalyzes:

glyceraldehyde-3-phosphate + NAD⁺ + P_i  → 1,3,bisphosphoglycerate + NADH + H⁺

This is the only step in Glycolysis in which NAD⁺ is reduced to NADH

A cysteine thiol at the active site of Glyceraldehyde-3-phosphate Dehydrogenase has a role in catalysis . 

7. Phosphoglycerate Kinase catalyzes:

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

This transfer of phosphate to ADP, from the carboxyl group on 1,3-bisphosphoglycerate, is reversible

8. Phosphoglycerate Mutase catalyzes:  3-phosphoglycerate → 2-phosphoglycerate

Phosphate is shifted from the hydroxyl on C3 of 3-phosphoglycerate to the hydroxyl on C2.  

9. Enolase catalyzes:  2-phosphoglycerate  → phosphoenolpyruvate + H₂O

This Mg⁺⁺-dependent dehydration reaction is inhibited by fluoride. Fluorophosphate forms a complex with Mg⁺⁺ at the active site .

10. Pyruvate Kinase catalyzes:  phosphoenolpyruvate + ADP  → pyruvate + ATP

This transfer of phosphate from PEP to ADP is spontaneous. 

Balance sheet for high energy bonds of ATP: 

• 2 ATP expended
• 4 ATP produced (2 from each of two 3C fragments from glucose) 
• Net Production of 2~ P bonds of ATP per glucose

Classification of Fatty Acids and Triglycerides

Short-chain: 2-4 carbon atoms

Medium-chain: 6-12 carbon atoms

Long-chain: 14-20 carbon atoms

Very long-chain: >20 carbon atoms

• are usually in esterified form as major components of other lipids

A16-carbon fatty acid, with one cis double bond between carbon atoms 9 and 10 may be represented as 16:1 cisD⁹.

Double bonds in fatty acids usually have the cis configuration. Most naturally occurring fatty acids have an even number of carbon atoms

Examples of fatty acids

There is free rotation about C-C bonds in the fatty acid hydrocarbon, except where there is a double bond. Each cis double bond causes a kink in the chain,

ZINC

The enzyme RNA polymerase, which is required for transcription, contains zinc and it is essential for protein bio synthesis.

Deficiency in Zinc leads to poor wound healing, lesions of skin impaired spermatogenesis, hyperkeratosis, dermatitis and alopecia

K_eq, K_w and pH

As H₂O is the medium of biological systems one must consider the role of this molecule in the dissociation of ions from biological molecules. Water is essentially a neutral molecule but will ionize to a small degree. This can be described by a simple equilibrium equation:

H₂O <-------> H⁺ + OH^-

This equilibrium can be calculated as for any reaction:

K_eq = [H⁺][OH^-]/[H₂O]

Since the concentration of H₂O is very high (55.5M) relative to that of the [H⁺] and [OH^-], consideration of it is generally removed from the equation by multiplying both sides by 55.5 yielding a new term, K_w:

K_w = [H⁺][OH^-]

This term is referred to as the ion product. In pure water, to which no acids or bases have been added:

K_w = 1 x 10^-14 M²

As K_w is constant, if one considers the case of pure water to which no acids or bases have been added:

[H⁺] = [OH^-] = 1 x 10^-7 M

This term can be reduced to reflect the hydrogen ion concentration of any solution. This is termed the pH, where:

pH = -log[H⁺]