Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438 - Introductory Biochemistry - Spring 2013

Lecture Notes:

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beta-oxidation of Fatty Acids

Free fatty acids are introduced into the cytosol, but Beta-oxidation occurs in the mitosol. Two situations occur:

Short to medium length fatty acids are permeable to the mitochondrial membrane. They are activated to fatty acyl CoA derivatives in the mitochondrial matrix by Butyryl-CoA Synthetase:

Butyryl-CoA Synthetase reaction diagram

Note that two ATP equivalents are required: the phosphoanhydride and thioester bonds are of similar free energies, so a second phosphoanhydride bond is also hydrolyzed to drive the reaction to completion.

Long chain fatty acids

are bound to Fatty acid binding protein for transport within the cytosol. They are impermeable to the inner mitochondrial membrane (they are also toxic to the mito!). They are thus esterified in the cytosol by microsomal Fatty acyl CoA synthetase in a reaction identical to the one shown above. Again the reaction is driven by the hydrolysis of pyrophosphate. The enzyme involves an acyl AMP intermediate:

Fatty acyl CoA synthetase reaction mechanism

with Ping Pong Bi Uni-Uni Bi kinetics:

Fatty acyl CoA synthetase kineticmechanism diagram

Carnitine Carrier

The resulting acyl CoA ester is still not permeable to the mitochondrial membrane so a carrier system is needed. In this system the fatty acyl group is transferred from CoA-SH to carnitine, diffuses across the membrane, and then transferred back to another CoA-SH within the matrix:

diagram of the Carnitine Carrier reactions

The carnitine transport step across the inner membrane is the slow step and flux generating step for Beta-oxidation of long chain fatty acids. Note that this system maintains separate pools of CoASH in the cytosol vs. the matrix.

Beta-oxidation

Once inside the mitochodrial matrix fatty acyl CoA can be broken down in the matrix by the fatty acid Beta-oxidation cycle [slide] as shown in Figure 12.8, and the Beta-oxidation scheme in your Biochemistry Packets. Note that the first three reactions of Beta-oxidation are the "mainline sequence" reactions we've already seen in the TCA Cycle. So you already know nearly all the reactions! The last reaction of the cycle releases an acetyl-CoA via a Claisen cleavage reaction (like an aldol cleavage but for esters instead of aldehydes). Note the similarity to the Claisen condensation from organic chemistry:

Claisen condensation mechjanism diagram

but of course run in reverse, and with CoAS- substituting for the alkoxide ion in the cleavage reaction.

Energy Yield

If we calculate the energy production for the complete oxidation of palmitate (16 C 's) we get:

 Reaction Energy Product Factor Multiplier ATP Equiv.

 O's
FACoA Syn. AMP -2 1 -2
Flavin DH FADH2 1.5 (2) 7 10.5 (14)
NAD+ DH NADH 2.5 (3) 7 17.5 (21)  7

8 AcCoA's to Kreb's TCA Cycle
NADH 2.5 (3) x 3 8 60 (72)  24
GTP 1 8 8  0
FADH2 1.5 (2) 8 12 (16)  8
Total       106 (129)  46

If we look at ATP/C we get 106/16= 6.63, while for glucose we get 32/6= 5.33, and for hexanoate: 36/6= 6. Thus, as expected, the fatty acids, being more reduced on average, give more energy per carbon and per gram. Along with the fact that they are stored without water of hydration, unlike carbohydrates, we can see their advantage as energy storage molecules for mobile organisms.

Another measure of fuel use is the P/O ratio, the number of ATP's generated for each oxygen atom consumed. For palmitate P/O = 106/46 = 2.3. As a comparison the P/O for glucose = 32/12 = 2.67. Notice by this measure glucose is the better fuel in situation where oxygen is limiting, since glucose will give more ATP's per mL of oxygen.

 

beta-OXIDATION OF UNSATURATED FATTY ACIDS

One might think that since an unsaturated fatty acid is created during beta-oxidation that unsaturated fatty acids should be handled easily by this system. However two problems occur due to the high level of specificity of the enzymes involved:

Two new enzymes are required to handle these situations: Enoyl-CoA isomerase (isomerizes a cis-3,4-double bond to a trans-2,3-double bond), and 2,4-Dienoyl-CoA reductase (reduces the cis-4,5-double bond in the trans-2,3-cis-4,5-dienoyl-CoA derivative formed during beta-oxidation). The resulting products are then broken down by the beta-oxidation enzymes.

ODD-CHAIN FATTY ACIDS

Most biological fatty acids are of even-numbered carbon chains. However, some organisms, particularly in the arctic marine environment, have a relatively high odd-chain component. Thus in organisms such as traditional Eskimo (Innuit) and polar bears eating lots of seal blubber and fish, odd-chain fatty acids can constitute a significant dietary component. These fatty acids are handled normally through beta-oxidation until the last turn, where pentyl-CoA is cleaved into acetyl-CoA and propionyl-CoA. The propionyl-CoA is converted through a number of steps to succinyl-CoA. These steps involve addition of carbon dioxide (with ATP energy) and an isomerization requiring cobalamin derived from vitamin B12. The succinyl-CoA can then be metabolized normally via the TCA cycle to malate, then to PEP and then to either 2-PGA for gluconeogenesis or to Pyruvate for energy production. (Propionate metabolism is also important to ruminants, since it is produced as a fermentation product by their symbiotic bacteria from plant matter.)

Ketone Bodies

Introductory discusion of fat metabolism, exercise, and fasting.

Fatty acids can be used as the major fuel for tissues such as muscle, but they cannot cross the blood-brain barrier, and thus cannot be used by the central nervous system (CNS). This becomes a major problem during starvation (fasting), particularly for organisms such as ourselves in which CNS metabolism constitute a major portion of the resting basal metabolic rate. These organism must provide glucose to the CNS to provide for metabolic needs, and thus during the initial fasting period must break down substantial amounts of muscle tissue (protein) to provide the amino acid precursors of gluconeogenesis. Obviously the organism could not survive long under such a regime. What is needed is an alternate fuel source based on fat rather than muscle. The so-called ketone bodies serve this function:

ketone body structures

Note that only two of the ketone bodies are in fact ketones, and that acetone is an "unintentional" breakdown product resulting from the instability of acetoacetate at body temperature. Acetone is not available as fuel to any significant extent, and is thus a waste product.

CNS tissues can use ketone bodies any time, the problem is the normally very low concentrations (< 0.3 mM) compared to glucose (about 5 mM). Since the KM's for both are similar, the CNS doesn't begin to use ketone bodies in preference to glucose until their concentration exceed's the concentration of glucose in the serum.

The system becomes saturated at about 7 mM. The limiting factor in using ketone bodies then becomes the ability of the liver to synthesis them, which requires the induction of the enzymes required for acetoacetate biosynthesis. Normal glucose concentrations inhibit ketone body synthesis, thus the ketone bodies will only begin to be synthesized in high concentrations as serum glucose concentrations fall. As an example, ketone bodies might start at about 0.1 mM after an overnight fast, rise to 3 mM after a 3 day fast, and go to 7-8 mM with prolonged fasting (>24 days).

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© R. A. Paselk 2010;

Last modified 19 April 2013