Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2007
Lecture Notes: 3 December	© R. Paselk 2007
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KETONE BODIES, cont.

Ketogenesis

The so-called ketone bodies are:

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 (lost through respiration etc.).

 

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%

Mitochondrial Electron Transport

 

Mitochondria: First let's review mitochondrial structure (Figure 14.2) [overhead 14.6a H, 15.2 MvH]. Recall that the inner membrane is very tight - that is the passage of polar molecules and ions is prevented without a specific transport vehicle.

ENERGY AND REDUCTION POTENTIALS

The electron transport system involves a variety of redox reactions, so it is useful to review some electrochemical relationships. First, the free energy of a reaction is related to the reduction potential for electron transfer by the equation:

G°' = - nFE°'

where n= moles of electrons transferred, and F is Faraday's constant (96,485 J V^-1mol^-1). The standard reduction potential for a reaction can be found from the difference between the reduction potentials of the electron acceptors and donors:

E°'= E°'_{e^- acceptor}- E°'_{e^- donor}

(Table 19-2 in your text lists biologically useful reduction potentials. Note that the higher the positive value of the reduction potential, the greater the tendency to pick up electrons: that is, electrons flow from negative to positive reduction potentials. The potentials are all relative to the potential of the SHE, or Standard Hydrogen Electrode, with a defined potential of Zero V.) The Nernst equation, which describes the reduction potential for an electrochemical reaction,

E= E° '- (RT / nF) lnQ

is very similar to the free energy equation,

G = G°' + RTlnQ,

while the equation for the reduction potential for an equilibrium system,

E°' = (RT / nF) lnK_eq,

reminds one of the free energy/equilibrium relationship,

G°' = - RTlnK_eq.

Note the standard reduction potentials and resultant standard free energies: NADH: -0.315 V; O₂: +0.815 V. So for the reaction:

1/2 O₂ + NADH H₂O + NAD⁺

we have 0.815 - (- 0.315) = 1.130 V, which, using the relationship between free energy and potential gives -218 kJ/mol. The free energy of hydrolysis of ATP is -30.5 kJ/mol, so if three ATP are made/pair of electrons flowing through ETS 91.5 kJ are captured out of 218 kJ available, or 42% . This is of course under "Standard Conditions." It is thought by some that under physiological conditions this may actually be closer to 70%.

THE ELECTRON TRANSPORT SYSTEM

The inner mitochondrial membrane is protein rich. If carefully broken down we find it is very rich in five protein complexes: I -IV are large protein complexes involved in electron transport, while V is the ATP sythatase driven by proton gradients. It turns out that if you grind up mitochondria carefully the four complexes can be isolated from the inner membrane that are involved in electron transport. These complexes appear to be independently "floating" in the inner membrane. Some of the properties for mammalian complexes are listed in the table:

modified from Moran & Scrimgeour Biochemistry 2nd ed. (1994) p 18.8 updated with Matti Saraste "Oxidative Phosphorylation at the fin de siécle " Science 283 (5 March 1999) pp 1488-1492.  Complex MW Subunits Redox Components I. NADH:ubiquinone (CoQ) oxidoreductase 900+ kD ( 'L' shaped complex) 42 or 43 subunits of unknown stoichiometry (25-26 types) 1 FMN; 7 or 8 different Fe-S centers, covalently bound lipid, 3 or more bound quinols. II. Succinate:ubiquinone (CoQ) reductase 127 kD (anchored to membrane by b cytochrome) 4 1 FAD; 3 Fe-S clusters; 1 cytochrome b 560  III. CoQ:cytochrome c reductase (Cytochrome bc1) 280 kD 11 (only 3 of which have redox centers and bacterial homologs)  2 cytochrome b; 1 Fe-S cluster; 1 cytochrome c 1 IV. Cytochrome c oxidase 400 kD dimer (13 chains each) 1 cytochrome a ; 1 CuA; 1 cytochrome a 3; 1 CuB; bound phospholipids

Electron flow through the ETS is summarized below (see also Figure 14.6). [overhead] Only major electron carriers are shown within complexes (enclosed in brackets):

NADH [FMN Fe-S] Q [Fe-S/Cyt b Cyt c₁] Cyt c [Cyt a Cyt a₃] O₂

Diagramatically:

Notice the central position of Coenzyme Q (aka ubiquinone, CoQ₁₀). The pool of CoQ forms a reservoir where reducing equivalents may be stored between the various inputs to the ETS, such as Complex I or Complex II and Complex III [overhead].

Pathway Diagrams

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Last modified 3 December 2007