Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438 - Introductory Biochemistry - Spring 2013

Lecture Notes:

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Ketogenesis

Ketogenesis occurs in the matrix of liver mitochondria. Fatty acids are first broken down to acetyl CoA via beta-oxidation (providing energy for liver metabolism from the reducing equivalents generated). The acetyl CoA is then used in ketogenesis:

Biosynthesis reactions for ketone body synthesis in teh liver

Depending on the status of the liver, acetoacetate can now be reduced to give D-beta-hydroxybutyrate, which delivers more reducing equivalents, and thus ATP equivalents, to the peripheral tissues at the expense of the liver:

reduction of acetoacetate to give D-beta-hydroxybutyrate

Ketone Bodies as Fuel

The ketone bodies are water soluble and are transported across the inner mitochondrial membrane as well as across the blood-brain barrier and cell membranes. Thus they can be used as a fuel source by a variety of tissues including the CNS. They are preferred substrates for aerobic muscle and heart, thus sparing glucose when they are available.

In the peripheral tissues the ketones must be reconverted to acetyl CoA in the mitochondria:

oxidation of D-beta-hydroxybutyrate in the peripheral tissues

The energy provided to the peripheral tissues from acetoacetate and for beta-hydroxybutyrate are shown below:
Reaction Energy Product Factor Multiplier ATP Equiv.
CoA transferase succinate (-GTP) 1 1 -1
Kreb's NAD+ DH's NADH 2.5 x 3 2 15
Kreb's GTP 1 2 2
Kreb's FADH DH FADH2 1.5 2 3

Acetoacetate Total:

19
Butyrate DH NADH 2.5 1 2.5

Butyrate Total:

 21.5

 

Note the P/O ratios for the ketone bodies: Acetoacetate = 19 ATP / 8 O = 2.38; Butyrate = 21.5 ATP / 9 O = 2.39 which are higher than we calculated for palmitate (2.3), but again lower than for glucose (2.67).

These reactions can be thought of as giving the liver overall control of fat metabolism. Lower vertebrates store fat in the liver. In a sense adipose tissue can be thought of then as "extended liver" tissue metabolically. Note that the liver can adjust the amount of reducing equivalents, and thus ATP equivalents, it sends to the peripheral tissues by adjusting the amounts of acetoacetate vs. beta-hydroxybutyrate it exports. Thus the percentage of the free energy distributed between the tissues is shown below:

 Compound \ Tissue Liver Peripheral Tissues
3-hydroxybutyrate 17% 83%
Acetoacetate 26% 74%

Fatty Acid Biosynthesis

[Reference: Wakil, S. J., Stoops, J. K., and Joshi, V. C., Ann. Rev. Biochem. 52, 537-79 (1983).]

Chemically fatty acid biosynthesis is a review of of beta-oxidation and pyruvate carboxylation. Want to reverse the reactions of beta-oxidation. But beta-oxidation is highly favorable as is, and want synthesis to also be favorable - obviously some variations in the pathways are needed.

Two reactions enable the synthesis pathway:

The first step in fatty acid biosynthesis is to activate acetyl-CoA by the addition of a carbon dioxide using Acetyl-CoA carboxylase. This reaction is chemically identical to the Pyruvate carboxylase reaction seen earlier (Lecture 23, 25 March, Gluconeogenesis):

Acetyl-CoA carboxylase reaction

This reaction is physiologically irreversible and is the flux generating or first committed step of fatty acid biosynthesis. As expected it is regulated. In mammals acetyl-CoA carboxylase is a large enzyme existing as inactive protomers (560,000 MW, 4 subunits, one biotin), which can assemble into active filaments (4 - 10 million MW).

Formation of the filaments (activation) is

As we shall see these are excellent indicators of the fuel status of the cell.

In addition to the activator/inhibitor controls of citrate and fatty acyl-CoA, the enzyme is also under hormonal control (note the similarities to glycogen control):

In mammals Fatty Acid Synthase (FAS) [slide] catalyzes fatty acid synthesis on a homodimeric enzyme, each monomer of which has seven catalytic activities, and eight sites! (In bacteria such as E. coli there are seven separate enzymes plus an acyl-carrier protein. Plants also have individual proteins for the various activities which are associated in a quaternary complex. In metazoans and fungi FASs are complexes of multifunctional proteins. Note: the numbers in the sites correspond to the numbered reactions of the FA Synthase reactions diagram, below.) The enzyme weighs approximately 500,000 Daltons. [Reference: Maier Timm, Simon Jenni and Nenad Ban. "Architecture of Mammalian Fatty Acid Synthase at 4.5 A Resolution." Science 311(3 March 2006) 1258; mini review comparing fungal and mammalian enzymes - Smith, Stuart. "Architectural Options for a Fatty Acid Synthase." Science 311(3 March 2006) 1251.]

Fatty Acid Biosynthesis, cont.

[Reference: Wakil, S. J., Stoops, J. K., and Joshi, V. C., Ann. Rev. Biochem. 52, 537-79 (1983).]

Fatty Acid Synthase (FAS)

There are two carriers on this complex.

Looking at the the Fatty Acid Synthase reactions [slide] (packet or Figure 16.5):
We now have a beta-ketoacyl group ready to go through the reverse of the reactions of beta-oxidation.

Pyruvate-Malate Cycle

The reactions of fatty acid synthesis all take place in the cytosol, but acetyl-CoA is made in the mitochondria and can't cross the inner membrane. The Pyruvate-Malate Cycle (Citrate-Pyruvate Cycle) [slide] (packet) is used to take acetyl- groups to the cytosol while simultaneously providing a source of NADPH from NADH, and thus coupling fatty acid synthesis to Glycolysis. Note that the acetyl-CoA is first joined to oxaloacetate to make citrate which is readily transported out of the mitochondria using a co-transporter. The citrate is then cleaved to acetyl-CoA and oxaloacetate, a process requiring ATP to make it favorable (recall the condensation was spontaneous). Acetyl-CoA for fatty acid synthesis is now available in the cytosol, but oxaloacetate must be regenerated for the mitosol.

The cytosolic oxaloacetate is now dehydrogenated to give malate and NAD+. Malate is next oxidized by Malic enzyme to give pyruvate in a reaction which also provides NADPH for use in biosynthesis. (Note that NADH generated in Glycolysis is "converted" to NADPH for F.A. synthesis in these two reactions, while simultaneously regenerating the NAD+ needed to continue Glycolysis!) The pyruvate can now cross into the mitosol to be used in regenerating oxaloacetate.

Now we have all of the pieces of fatty acid biosynthesis starting from glucose. Let's look at the integration of the various pathways involved: Glycolysis, hexose monophosphate shunt, Pyruvate-malate shuttle, and Fatty acid biosynthesis. Notice the provision of reducing equivalents, redox balance and provision of required cytosolic ATP's as well as carbon source.

Elongation of Fatty Acids

If the Fatty Acid Synthetase Complex only makes palmitate where do the rest of the fatty acids come from? Of course palmitate can be shortened by beta-oxidation. For longer fatty acids there is a fatty acid elongation system localized on the ER. The same reactions occur as in the Synthetase, but now have individual enzymes. Palmitate is first activated to palmitoyl-CoA. The enzymes prefer C-16 or less as substrate; thus the major product is stearoyl-CoA. However longer unsaturated fatty acids will also bind (the kinking of the cis double bond makes them effectively shorter), so unsaturated fatty acids of 20, 22, and 24-C's are also made. Thus most longer fatty acids are unsaturated.
 
A second system for fatty acid elongation exists in the mitosol, probably for provision of long fatty acids for mitochondrial structure. This system uses most of the same activities of beta-oxidation, but an NADPH dependent Enoyl-CoA reductase replaces the FAD dependent dehydrogenase.

Fatty Acid Desaturation

Plants and animals differ in where double bonds are introduced into fatty acids.

Plants put in Delta9 (a 9-10 double bond) and then can put in double bonds at three carbon intervals towards the tail (Delta12, Delta15). They can also add Delta6, but not common.

Animals also start with Delta9, then can add at three carbon intervals toward carboxy end (Delta6 and Delta3). Animals cannot add towards the tail. Therefore animals cannot make linoleoyl-CoA (Delta9, 12; C18) but can make oleoyl-CoA (Delta9; C18). Animals must therefore take in plant products (either directly as herbivores, or indirectly by eating herbivores) to acquire essential unsaturated fatty acids such as linoleic and arachadonic acid.

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Last modified 22 April 2013