Oxidation of Lipids II

Suaib Luqman

epgp books
 

 

 

  • Objectives

v To understand the oxidation of lipids

v How fatty acid oxidation takes place

 

  1. Concept Map
  1. Description

 

Mobilization and Transport of Fats: Role of Hormones

 

When the hormones indicate the call for the metabolic energy, TAGs accumulated in the adipose tissues are mobilized (fetch out of storage) and transported to the tissues (heart, renal cortex, skeletal muscle) in which fatty acids is oxidized for the production of energy. In response to the low blood glucose levels, the hormones (epinephrine and glucagon) secretion increases and triggers the activity of the enzyme Adenylyl cyclase in the plasma membrane of the adipocyte which produces cyclic AMP (the intracellular second messenger). Cyclic AMP-dependent Protein kinase A (PKA) phosphorylates perilipin A which allows lipase (hormone-sensitive) resides in the cytosol to shift to the surface of the lipid droplet, where it commences hydrolyzing TAGs to glycerol and fatty acids. PKA also phosphorylates lipase (hormone-sensitive), increasing its activity (2-3 times), but >50-fold increase in fat mobilization triggered by epinephrine is primarily due to perilipin phosphorylation. Cells with malfunctioning perilipin genes have almost no retort to increase the cAMP concentration; their lipase (hormone-sensitive) does not associate with lipid droplets.

 

Knoop Concept for β-Oxidation of Fatty Acid

 

 

The earliest theory on fatty acid oxidation was the ‘β-oxidation hypotheses’ of Franz Knoop in 1904. He hypothesized that the fatty acid chain is degraded in a step-wise fashion by the repeated removal of the two carbon atoms due to the oxidation at the β carbon atom. For example stearic acid (18 C chain,) would first be oxidized to the palmitic acid (16 C chain) and after that to 14 C chain ultimately to CO2 and water. Many researchers have added knowledge to the fatty acid oxidation mechanism but the fundamental concept given by Knoop is still valid.

 

The β-oxidation of fatty acids (long chain) is vital to the prerequisite of the organism energy and is of fastidious substance for skeletal and cardiac muscle. Yet, other organs principally the liver, but also the white adipose tissues, small intestine and kidney can exploit the β-oxidation products for the formation of ketone bodies which can, in turn, be utilized by other tissues for the energy. The association of lipid or fat oxidation with the deployment of carbohydrate as an energy source is complex and relies on development of tissues, exercise, nutritional state and a number of other factors such as infection and further pathological states.

 

Knoop’s Experiments: Mystery of Product of Odd and Even Fatty Acid

 

Mechanism of fatty acid oxidation as elucidated by Knoop was stated to be the oxidation at the β-carbon of fatty acids.

 

He did some basic biochemical experiments on dogs that eventually proved his point. The dogs were fed with straight chain fatty acids and through their ω-carbon atom was joined a phenyl group which served as reporter molecule as it was excreted and was not metabolized. Knoop examined the urine sample of these dogs and found out that when these dogs were given phenylbutyrate or even numbered carbon fatty acid they excreted phenylacetic acid or its derivative while when they were given phenylpropionate or odd numbered carbon fatty acid they eliminated benzoic acid or its derivative.

 

This experiment was repeatedly done with different fatty acid and ultimately he finalized his results as the degradation of fatty acids takes place by β-oxidation. As his experiments were based on synthetic fatty acid (fatty acids with phenyl residues in place of the terminal methyl groups) for elucidating the exact mechanism , the experiment became a landmark in biochemistry research.

 

This was the major breakthrough in the biochemistry that highlighted the importance of β-carbon in the fatty acids that is prone to cleavage yielding a two carbon unit fragment after the full degradation of the free fatty acids.

 

Experimental Details

 

  1. Feeding dogs with odd-numbered carbon fatty acid (a): Phenylpropionic acid (C6H5-CH2-CH2-COOH)

 

(b):    Phenylvaleric acid (C6H5-CH2-CH2-CH2-CH2-COOH)

 

Isolation from urine: Hippuric acid (C6H5-CO-NH-CH2-COOH) which is the conjugate of glycine and benzoic acid

 

  1. Feeding dogs with even-numbered carbon fatty acid

(a):    Phenylbutyric acid (C6H5-CH2-CH2-CH2-COOH) Isolation from urine: Phenylaceturic acid (C6H5-CH2-CO-NH-CH2-COOH)

 

 

The experimental observation guide Knoop to put forward his findings that the oxidation occurs at 3rd carbon which is the β-carbon resulting into β-keto acids which acquiesce fatty acids reduced by two carbon units. The results were further confirmed by Dakin who executed parallel experiments with phenylpropionic acid and isolated benzoylacetic acid (C6H5-CO-CH2-COOH), phenyl-β-hydroxypropionic acid (C6H5-CHOH-CH2-COOH), phenylacrylic acid (C6H5-CH=CH-COOH) along with hippuric acid. At the identical time, Embden and coworkers established that fatty acids (unsubstituted) are metabolized to ketone bodies by β-oxidation in perfused livers.

 

Thus, within 6 years from 1904-1910, basic strategy for fatty acid oxidation was established and after a 30-year lag period, again experiments in cell free system confirmed and elucidated whole mechanism of β -oxidation  through the experiments of Munoz and Leloir (1943) and Lehninger (1944). One important aspect through the work of Lehninger unveiled the energy ‘spark’ in the form of ATP which is required in β-oxidation.

 

Fatty acids combined with CoA through thioester linkage to form activated fatty acids were shown by Wakil and Mahler as well as by Kornberg and Pricer and it was based on previous studies of Lynen and coworkers who established the structure of acetate in active form to be Acetyl CoA as well as the work of Lipmann and coworkers who isolated and exemplify coenzyme A. Kennedy and Lehninger established sub-cellular location which is mitochondria of the β-oxidation system. Another proof came from the fact that citric acid cycle is also present in mitochondria. The mitochondrial location of this pathway contracted with the experiential coupling of fatty acid oxidation to TCA cycle and to the oxidative phosphorylation. Ochoa in New York, Lynen in Munich, and Green in Wisconsin during 1950s carried out the enzymatic reactions which was possible by the development of protein purification and spectrophotometric enzyme assays.

 

Major Simplified Steps of β-Oxidation of Fatty Acids

 

  1. The first step of the pathway is the ‘activation’ of the fatty acid by the formation of the coenzyme A derivative which is ATP-mediated.
  2. Next step is the de-saturation between α & β-carbon atoms, the active fatty acid are then oxidized by β-oxidation.
  3. Finally, formation of one mole of acetyl-CoA (active acetate) and a fatty acid shorter by two carbon atoms and because of a thiolytic split with CoA already activated for repetition of the oxidative cycle as shown below.

In 1949, Kennedy, E. and Lehninger, A. established that oxidation of fatty acids occur in mitochondria. Further work proved that the fatty acids get activated before they are able to enter the matrix of mitochondria. Thus, the activation takes place on the outer membrane of the mitochondria.

 

The step requires energy and thus ATP helps in the thioester linkage formation between SH group of CoA and carboxylic group of fatty acid.

 

Enzyme responsible for catalyzing this reversible reaction is known as fatty acid thiokinase or Acyl CoA synthetase or Fatty acid CoA ligase. This enzyme is present both inside and on the outer membrane of mitochondria as well as in endoplasmic reticulum and peroxisomes.

 

Detailed experiment by Paul Berg proved that this reaction can be divided into two individual steps. In first reaction, Acyl adenylate is formed when fatty acid (carboxylic group) reacts with ATP (phosphoryl group). Activation by adenylation is something common as it occurs in activation of amino acids also; this is an example of convergent evolution.

 

The remaining two phosphoryl group is released as pyrophosphate (PPi) which is swiftly broken down by pyrophosphatase helping in converting the reversible reaction to forward driven reaction. The next reaction is the attack of CoA on Acyl adenylate to form AMP and Acyl CoA.

 

β-oxidation of activated fatty acid in the mitochondrial matrix

 

After the activation of fatty acids on the outer membrane of the mitochondria, next problem faced by them is the transport to the matrix of mitochondria crossing the mitochondrial membrane (outer and inner). This is done through the help of carnitine [(3-hydroxy-4-trimethyl ammonium butyrate)] which is a zwitter ionic alcohol by the mode of conjugation reaction catalyzed by carnitine palmitoyl transferase I (also known as carnitine acyl transferase I). This enzyme is bound to the outer mitochondrial membrane and it catalyzes the reaction in which acyl carnitine is formed through transfer of acyl group to the hydroxyl group of carnitine from the sulfur atom of CoA.

 

 

Because of zwitter-ionic property of carnitine, acyl carnitine is able to penetrate the inner mitochondrial membrane. A shuttle system at the inner mitochondrial membrane contains the enzyme which is a membrane exchange transporter known as Carnitine-acylcarnitine translocase helps in transfer of acyl carnitine inside per one carnitine outside the matrix. Acyl carnitine reacts with CoA and results in the formation of acyl-CoA and carnitine is liberated catalyzed by carnitine palmitoyl transferase II (or known as carnitine acyltransferase II).

 

The muscle, heart, and kidney are primarily affected with diseases linked with carnitine and the enzyme involved in transfer of acyl fatty acid which ranges from severe weakness to mild muscle cramping and sometimes even death.

Several enzymes collectively called ‘fatty acid oxidase’ are found adjacent to the inner mitochondrial membrane or the respiratory chain in the mitochondrial matrix. Overall, the main reaction is oxidation of Acyl CoA to Acetyl CoA. These reactions are done repeatedly shortening the chain by two carbons in each cycle ultimately resulting into Acetyl CoA and each cycle generates FADH2 and NADH

 

  1. Summary

 

In this lecture we learnt about:

 

  • The oxidation of lipids or fats including Knoop’s Concept
  • The implications of the reactions involved in β-Oxidation

 

you can view video on Oxidation of Lipids II

Weblinks

 

 

Books

 

  1. Lehninger Principles of Biochemistry by David L. Nelson, Albert‎ L. Lehninger, Michael‎ M. Cox. 2008. https://books.google.co.in/books?isbn=071677108X
  2. Beta-oxidation of Unsaturated Fatty Acids in Yeast. 2007. Page 1 https://books.google.co.in/books?isbn=0549355103
  3. Incomplete Beta Oxidation of Unsaturated Fatty Acids. 2008. https://books.google.co.in/books?isbn=0549434305
  4. Textbook of Biochemistry for Medical Students by D M Vasudevan, Sreekumari‎ S, Kannan‎ Vaidyanathan. 2010. Page 133 https://books.google.co.in/books?isbn=9350250160
  5. Biochemistry by Richard A. Harvey (Ph. D.), Richard‎ A. Harvey, Denise‎ R. Ferrier. 2011. Page 191 https://books.google.co.in/books?isbn=160831412X
  6. Medical Sciences by Jeannette Naish, Denise‎ Syndercombe Court. 2014. Page 94 https://books.google.co.in/books?isbn=0702052493
  7. Carnitine Metabolism and Human Nutrition by Benjamin T. Wall, Craig‎ Porter. 2014. Page 106 https://books.google.co.in/books?isbn=1466554266