3-hydroxybutyrate transfer

The steps in the subsequent utilization of muscle LCFAs may be summarized as follows. The free fatty acids, liberated from triglycerides by a neutral triglyceride lipase, are activated to form acyl CoAs by the mediation of LCFAcyl-CoA synthetase which is situated on the outer mitochondrial membrane. The next step involves carnitine palmitoyl transferase I (CPT I, see Figure 9) which is also located on the outer mitochondrial membrane and catalyzes the transfer of LCFAcyl residues from CoA to carnitine (y-trimethyl-amino-P-hydroxybutyrate). LCFAcyl... 

At high concentrations of acetyl-CoA in the liver mitochondria, two molecules condense to form acetoacetyl CoA [1]. The transfer of another acetyl group [2] gives rise to 3-hydroxy-3-methylglutaryl-CoA (HMC CoA), which after release of acetyl CoA [3] yields free acetoacetate (Lynen cycle). Acetoacetate can be converted to 3-hydroxybutyrate by reduction [4], or can pass into acetone by nonenzymatic decarboxylation [5]. These three compounds are together referred to as "ketone bodies," although in fact 3-hydroxy-butyrate is not actually a ketone. As reaction [3] releases an ion, metabolic acidosis can occur as a result of increased ketone body synthesis (see p. 288). 

In extraliepatic tissues, d-/3-hydroxybutyrate is oxidized to acetoacetate by o-/3-hydroxybutyrate dehydrogenase (Fig. 17-19). The acetoacetate is activated to its coenzyme A ester by transfer of CoA from suc-cinyl-CoA, an intermediate of the citric acid cycle (see Fig. 16-7), in a reaction catalyzed by P-ketoacyl-CoA transferase. The acetoacetyl-CoA is then cleaved by thiolase to yield two acetyl-CoAs, which enter the citric acid cycle. Thus the ketone bodies are used as fuels. 

However, if the electron transport between 3-hydroxybutyrate and cytochrome b562 is tightly coupled to the synthesis of one molecule of ATP, the observed potential of the carrier will be determined not only by the imposed potential E of the equilibrating system but also by the phosphorylation state ratio of the adenylate system (Eq. 18-7). Here AG atp is the group transfer potential (-AG of hydrolysis) of ATP at pH 7 (Table 6-6), and n is the number of electrons passing through the chain required to synthesize one ATP. In the upper part of the equation n is the number of electrons required to reduce the carrier, namely one in the case of cytochrome b562. 

The first step in valine biosynthesis is a condensation between pyruvate and active acetaldehyde (probably hy-droxyethyl thiamine pyrophosphate) to yield a-acetolactate. The enzyme acetohydroxy acid synthase usually has a requirement for FAD, which, in contrast to most flavopro-teins, is rather loosely bound to the protein. The very same enzyme transfers the acetaldehyde group to a-ketobutyrate to yield a-aceto-a-hydroxybutyrate, an isoleucine precursor. Unlike pyruvate, the a-ketobutyrate is not a key intermediate of the central metabolic routes rather it is produced for a highly specific purpose by the action of a deaminase on L-threonine as shown in figure 21.10. 

The use of unprotected a-hydroxy acids may also be compatible with the preparation of depsipeptides. However, attempts to couple L-a-hydroxyisovaleric acid and benzyl phenyl-alaninate using TBTU resulted in the formation of considerable amounts of the hy-droxycarbamoylated compound,in contrast to the smooth reaction mediated by PyBOP. Transfer of the dimethylcarbamoyl moiety of uroniuno/guanidinium salts appears to be restricted to a-hydroxy acids and has not been observed in coupling reactions between Fmoc-Ser-OH, Fmoc-Thr-OH, or L-3-hydroxybutyric acid and H-Phe-OBzl in the presence of TBTU/NMM in... 

Acetoacetate is converted into acetyl CoA in two steps. First, acetoacetate is activated by the transfer of CoA from succinyl CoA in a reaction catalyzed by a specific CoA transferase. Second, acetoacetyl CoA is cleaved by thiolase to yield two molecules of acetyl CoA, which can then enter the citric acid cycle (Figure 22.22). The liver has acetoacetate available to supply to other organs because it lacks this particular CoA transferase. 3-Hydroxybutyrate requires an additional step to yield acetyl CoA. It is first oxidized to produce acetoacetate, which is processed as heretofore described, as well as NADH for use in oxidative phosphorylation. 

In muscle, most of the fatty acids undergoing beta oxidation are completely oxidized to C02 and water. In liver, however, there is another major fate for fatty acids this is the formation of ketone bodies, namely acetoacetate and b-hydroxybutyrate. The fatty acids must be transported into the mitochondrion for normal beta oxidation. This may be a limiting factor for beta oxidation in many tissues and ketone-body formation in the liver. The extramitochondrial fatty-acyl portion of fatty-acyl CoA can be transferred across the outer mitochondrial membrane to carnitine by carnitine palmitoyltransferase I (CPTI). This enzyme is located on the inner side of the outer mitochondrial membrane. The acylcarnitine is now located in mitochondrial intermembrane space. The fatty-acid portion of acylcarnitine is then transported across the inner mitochondrial membrane to coenzyme A to form fatty-acyl CoA in the mitochondrial matrix. This translocation is catalyzed by carnitine palmitoyltransferase II (CPTII Fig. 14.1), located on the inner side of the inner membrane. This later translocation is also facilitated by camitine-acylcamitine translocase, located in the inner mitochondrial membrane. The CPTI is inhibited by malonyl CoA, an intermediate of fatty-acid synthesis (see Chapter 15). This inhibition occurs in all tissues that oxidize fatty acids. The level of malonyl CoA varies among tissues and with various nutritional and hormonal conditions. The sensitivity of CPTI to malonyl CoA also varies among tissues and with nutritional and hormonal conditions, even within a given tissue. Thus, fatty-acid oxidation may be controlled by the activity and relative inhibition of CPTI. 

Acetolactate synthase can also transfer the two-carbon fragment from pyruvate to a-ketobutyrate, forming a-aceto-a-hydroxybutyrate. This is the first step in the formation of isoleucine. Propose a mechanism for this reaction. 

Madden LA, Anderson AJ, Asrar J, Berger P, Garrett P (2000) Production and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) synthesized by Ralstonia eutropha in fed-batch cultures. Polymer 41 3499-3505 Madden LA, Anderson AJ, Shah DT, Asrar J (1999) Chain termination in polyhydroxyalkanoate synthesis involvement of exogenous hydroxy-compounds as chain transfer agents. Int J Biol Macromol 25 43-53... 

Fig. 1 The effect of poly[(f )-3-hydroxybutyrate] (PHB) on survival capability of starved bacteria. Cells of Azospirillum brasilense Sp7 (filled triangles) and phaC mutant (filled circles) were grown on a medium with a high carbon to nitrogen ratio for 24 h and transferred to phosphate buffer, where they were incubated for 12 days. Bacterial density was determined using dilution plating (Reproduced from Kadouri et al. 2002, with kind permission from the American Society for Microbiology)...

Intracellular degradation (often called mobilization) consists in enzymatic breakdown of polymers to monomers, which are then converted by o-hydroxybutyrate dehydrogenase into acetacetate. As a result of the dehydrogenase reaction, the latter is transferred to CoA, serving as a substrate for P-ketothiolase, which converts it into acetyl-CoA. Studies of intracellular degradation may be important in regard to mass production of microbial polyesters. 

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