Chem 438 - Introductory Biochemistry - Spring 2013
Lecture Notes:
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:
- The first reaction (1), catalyzed by thiolase, involves a Claisen condensation of two acetyl CoA's (essentially a reversal of the last reaction of beta-oxidation) to give acetoacetyl CoA - almost the final product! The problem now is to remove the CoASH.
- This is done in a two stage process involving first the addition of a third acetyl CoA by the second enzyme: HMG-CoA synthase (2) via an aldol condensation (note the similarity to the formation of citrate in the TCA cycle, including driving the reaction by the hydrolysis of a thiol ester bond).
- The resulting beta-Hydroxy-beta-glutaryl-CoA is now cleaved by HMG-CoA lyase (3), regenerating acetyl CoA and giving the product: acetoacetate.
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:
-
-
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:
- If we start with beta-hydroxybutyrate, then it is first oxidized to acetoacetate with the production of one NADH (1).
- Coenzyme A must now be added to the acetoacetate. The thioester bond is a high energy bond, so ATP equivalents must be used. In this case the energy comes from a transesterification of the CoAS from succinyl CoA to acetoacetate by Coenzyme A transferase (2). The succinyl CoA comes from the TCA cycle where a GTP is not made.
- The acetoacetyl CoA is now cleaved to two acetyl CoA's with Thiolase (3).
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:
- Acetyl-CoA is "activated" by the addition of a carbon dioxide, and
- NADPH is substituted as a more powerful reducing agent than FADH2.
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):
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
- promoted by citrate, but
- inhibited by fatty acyl CoA.
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):
- glucagon stimulates phosphorylation and thus inactivation of the liver enzyme, while
- adrenalin (epinephrin) stimulates phosphorylation and inactivation in adipose tissue.
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).]
There are two carriers on this complex.
- The first is referred to as ACP1 which acts as a holding station for acetyl- or fatty acyl- groups. In either case they are bound to a cysteinyl sulfhydryl group.
- The second, ACP2, binds the growing fatty acyl chain during the condensation and reduction reactions of the cycle. In this case the acyl group is carried on a long phosphopantetheine prosthetic group. This arm allows the growing chain to move among the various active sites without being lost to the solution.
- Looking at the the Fatty Acid Synthase reactions [slide] (packet or Figure 16.5):
- the first reaction, catalyzed by Acetyl-CoA:ACP transacylase, transfers an acetyl group from Coenzyme A to the cysteinyl-S on ACP1.
- Next, a malonyl-group is transferred from a Coenzyme A to the pantetheinyl-S of ACP2 by Malonyl-CoA:ACP transacylase.
- Now the carbon dioxide leaves the malonyl group, with the electrons from its bond attacking the acyl group on ACP1. This reaction is catalyzed by Ketoacyl-ACP synthase.
- We now have a beta-ketoacyl group ready to go through the reverse of the reactions of beta-oxidation.
- Thus the keto-group is reduced to an alcohol using NADPH (beta-ketoacyl-ACP reductase),
- followed by the elimination of the alcohol (Enoyl-ACP hydrase) to give the cis-2,3-enoyl group.
- The enoyl is then reduced with NADPH substituting for FADH2 (Enoyl-ACP reductase) to give the saturated acyl group.
- Finally the acyl group is transferred from the pantotheinyl-S of ACP2 to the cysteinyl-S on ACP1 (ACP-acyltransferase) leaving ACP2 available to pick up the next malonyl moiety.
- After seven turns of the cycle palmitate is released.
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
-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 -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 
9 (a 9-10 double bond) and then can put in double bonds at three carbon intervals towards the tail (12, 15). They can also add 6, but not common.
Animals also start with 9, then can add at three carbon intervals toward carboxy end (6 and 3). Animals cannot add towards the tail. Therefore animals cannot make linoleoyl-CoA (9, 12; C18) but can make oleoyl-CoA (9; 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.
© R. A. Paselk 2010;
Last modified 22 April 2013
Pathway Diagrams
