2-aceto-2-hydroxybutyrate

The first committed step in the biosynthetic pathway of the branched chain amino acids is catalyzed by the enzyme acetohydroxyacid synthase (AHAS, EC 2.2.1.6), which is also referred to as acetolactate synthase (ALS). As depicted in Fig. 2.1.1, the pathway leading to valine and leucine begins with the condensation of two molecules of pyruvate accompanied by loss of carbon dioxide to give (S)-2-acetolactate. A parallel reaction leading to isoleucine involves the condensation of pyruvate with 2-ketobutyrate to afford (S)-2-aceto-2-hydroxybutyrate after loss of carbon dioxide. Both reactions are catalyzed by AHAS, which requires the cofactors thiamin diphosphate (ThDP) and flavin adenine dinudeotide (FAD). A divalent metal ion, most commonly is also required. Several excellent reviews... 

The other ketone bodies are derived from acetoacetate P-hydroxybutyrate, by reduction with the involvement of NAD-dependent hydroxybutyrate dehydrogenase, and acetone, by decarboxylation of acetoacetate with the participation of aceto-acetate decarboxylase ... 

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. 

Excess acetate (C2) can be converted to the mobile ketone body energy source aceto-acetate (C4) and thence its reduced form hydroxybutyrate (C,) for transport throughout the body. Excess acetate can be carboxylated (via acetylCoA carboxylase) to form malonylCoA (C3), the donor for further C2 additions (with C02 elimination) in the anabolic synthesis of long chain fatty acids. Fatty acids are components of the phospholipids of cellular membranes and are also stored as triacylglycerols (triglycerides) for subsequent hydrolysis and catabolic fatty acid oxidation to yield reduced coenzymes and thence ATP (see Chapter 2). 

Acetolactate syntnase (ALS) catalyses the reaction of two pyruvate molecules to give acetolactate and carbon dioxide. It also catalyses the reaction of pyruvate and a-ketobutyrate to give a-aceto-a-hydroxybutyrate and carbon dioxide. The enzyme requires three coenzymes for activity flavin adenine dinucleotide, thiamin pyrophosphate, and magnesium 1on. The reaction takes place in several steps. 

The syntheses of valine, leucine, and isoleucine from pyruvate are illustrated in Figure 14.9. Valine and isoleucine are synthesized in parallel pathways with the same four enzymes. Valine synthesis begins with the condensation of pyruvate with hydroxyethyl-TPP (a decarboxylation product of a pyruvate-thiamine pyrophosphate intermediate) catalyzed by acetohydroxy acid synthase. The a-acetolactate product is then reduced to form a,/3-dihydroxyisovalerate followed by a dehydration to a-ketoisovalerate. Valine is produced in a subsequent transamination reaction. (a-Ketoisovalerate is also a precursor of leucine.) Isoleucine synthesis also involves hydroxyethyl-TPP, which condenses with a-ketobutyrate to form a-aceto-a-hydroxybutyrate. (a-Ketobutyrate is derived from L-threonine in a deamination reaction catalyzed by threonine deaminase.) a,/3-Dihydroxy-/3-methylvalerate, the reduced product of a-aceto-a-hydroxybutyrate, subsequently loses an HzO molecule, thus forming a-keto-/kmethylvalerate. Isoleucine is then produced during a transamination reaction. In the first step of leucine biosynthesis from a-ketoisovalerate, acetyl-CoA donates a two-carbon unit. Leucine is formed after isomerization, reduction, and transamination. 

Acetohydroxyacid synthase (AHAS), formerly referred to as acetolactate synthase, is involved in the biosynthesis of branched-chain amino acids in plants and many microorganisms. " AHAS catalyzes the condensation of two molecules of pyruvate to form acetolactate and CO2, or the condensation of one molecule of pyruvate with one molecule of cr-ketobutyrate to form cr-aceto-cr-hydroxybutyrate and CO2 (Equation (24)). 

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. 

Chernysheva et al. were the first to have obtained evidence of inhibition from the product of the reaction of hydrogenation of ethyl aceto-acetate on Raney nickel catalyst modified with (2/ ,3R)-tartaric acid into ethyl (R)-(-)-3-hydroxybutyrate. Similar results were obtained also in the enantio-... 

L-Isoleucine originates from 2-oxobutyric acid, a threonine derivative and activated acetaldehyde (C 4) as outlined in Fig. 196. Both compounds condense to form tx-aceto-a-hydroxybutyric acid from which 2,3-dihydroxy-3-methyl-... 

The liver is exceptionally well equipped to oxidize fatty acids. The ketone bodies (acetone, hydroxybutyrate, and aceto acetic acid) are among the products of fatty acid oxidation. 

The concentration and associated ratio of the ketone bodies, aceto-acetate and 3-hydroxybutyrate, may also be helpful [15, 18, 19]. Ketosis and keto-aciduria are observed in certain patients with a mitochondrial disorder. A non-physiological increase of ketone bodies postprandially may be another indicator of a mitochondrial defect (Saudubray et al). Increased 3-hydroxybutyrate/acetoacetate ratio may suggest a defect in the respiratory chain in liver tissue. 

Ketone body (Section 20.4) One of the substances aceto-acetate, /3-hydroxybutyrate, or acetone resulting from amino acid catabolism. 

Fio. 7. Scheme of biosynthesis of valine and isoleucine. R tepresents a methyl group in valine and an ethyl group in isoleucine biosynthesis. I is pyruvate or a-keto-butyrate II is a-acetolactate or a-aceto-a-hydroxybutyrate HI is a-keto-/3-hydroxy> isovalerate or a-keto- -hydroxy- S-metbylvalerate IV is a,/ -dihydroxyisovalerate or a-0-dihydroxy- -methylvalerate V is a-ketoisovalerate or a-keto-d-methylvalerate and VI is valine or isoleucine. 

Shortly afterwards they proposed a similar mechanism for isoleucine biosynthesis by the condensation of a-ketobut3n ate and acetaldehyde to yield a-aceto-a-hydroxybutyrate followed by a migration of the ethyl groups (163) from C-2 of the butyrate moiety to C-1 of the acetaldehyde moiety (Fig. 7, II). 

Important evidence that a-acetolactate and a-aceto-a-hydroxybutyrate are precursors of valine and isoleucine, respectively, was obtained by Wagner et cd. 164). These investigators isolated and identified acetyl-methylcarbinol and acetylethylcarbinol from a mutant strain of Neurospora blocked in the biosynthesis of valine and isoleucine. The two carbinol compounds were obtained as pteridine derivatives. They would be formed by decarboxylation of acetolactate and aceto-a-hydroxybutyrate. That the latter compounds are the probable sources of the carbinols is indicated by the fact that Neurospora possesses an active decarboxylase which catalyzes the conversion of acetolactate and aceto-a-hydroxybutyrate to their respective carbinol. Furthermore, since the aceto compounds are 3-keto acids, they would be expected also to decarboxylate spontaneously. 

Support that a-aceto-a-hydroxybutyrate is on the pathway of isoleucine biosynthesis was obtained by Watanabe et al, (166), who prepared this ketol condensation product of a-ketobutyric acid and acetaldehyde and demonstrated that it was converted to L-isoleucine by cell-free extracts of baker s yeast. 

Following the ketol condensation to yield a-acetolactate and a-aceto-a-hydroxybutyrate the question arises as to whether the pinacol rearrangement occurs first to form a-keto-jS-hydroxyisovaleric acid and a-keto-i9-hydroxy- 8-methylvaleric acid (Fig. 7, III), or whether there is prior reduction. The evidence favoring pinacol rearrangement followed by reduction of the a-keto- 3-hydroxy acids is that enzymes have been found in Neurospora and E. coli which catalyze the reduction of synthetically prepared a-keto-(8-hydroxyisovaleric acid and a-keto-j8-hydroxy-/3-methyl-valeric acid to the corresponding a,/8-dihydroxy acids (166, 167). 

Subsequently, the enzymic conversion of a-acetolactate to a,6-dihydroxyisovalerate and a-aceto-a-hydroxybutyrate to a,/3-dihydroxy-jS-methylvalerate was shown by Wagner et al. 166) with extracts of Neurospora and by Umbarger 167) with extracts of appropriately blocked E. coU mutants. These acids were then shown to be utUized for valine and isoleucine formation. 

Lehninger showed that, in mitochondrial preparations, the oxidation of L-/3-hydroxybutyrate was coupled with the uptake of inorganic phosphate through DPN. As previously discussed, the phosphorylation occurs somewhere in the electron transport system. The maximum free energy change for the over-all conversion of /3-hydroxybutyrate to aceto-... 

AHAS catalyzes the first reaction in the biosynthetic pathway of the branched-chain amino acids. In particular, it catalyzes the ThDP-dependent synthesis of both 2-S-aceto-lactate 29 and 2-S-aceto-2-hydroxybutyrate by the coupling between pyruvate and pyruvate or 2-ketobutyrate. But AHAS was shown also to catalyze efficiently the chiral... 

Fig. 11.2 Total ion current chromatogram (Varian MAT 44 GC-MS) of organic acids extracted using ethyl acetate and diethyl ether from the urine of a patient with propionic acidaemia and separated as their trimethylsilyl derivatives on a 25 m SE-54 WCOT capillary column using temperature programming from 70°C to 220°C at 4 C min Peak identifications are 1, lactate 2, 3-hydroxypropionate 3, 3-hydroxybutyrate 4, 2-methyl-3-hydroxybutyrate 5, 3-hydroxyisovalerate 6, 3-hydroxy- -valerate 7, aceto-acetate 8 and 9, 2-methyl-3-hydroxyvalerate 10, 3-oxovalerate 11, 2-methyl-3-oxo-valerate (isomer 1) 12,2-methylacetoacetate 13,2-methyl-3-oxovalerate (isomer 2) 14 propionylglycine 15, glutarate 16, adipate 17, 5-hydroxymethyl-2-furoate 18, 2-hydroxyglutarate 19,3-hydroxy-3-methylglutarate 20,4-hydroxyphenylacetate 21 and 22, methylcitrate 23,4-hydroxyphenyl-lactate 24, palmitate. (Redrawn with modifications from Truscott et al., 1979)...

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