P-hydroxybutyrate dehydrogenase

The enzymes responsible for their metabolism, d-P-hydroxybutyrate dehydrogenase, acetoacetate-succinyl-CoA transferase and acetoacetyl-CoA-thiolase, are present in... 

In this test, p-hydroxybutyrate in the presence of NAD is converted by P-hydroxybutyrate dehydrogenase to acetoacetate, producing NADH. Diaphorase catalyzes the reduction of nitroblue tetrazolium (NBT) by NADH to produce a purple compound and its absorbance is read in a special meter that provides a digital readout. 

The answer is e. (Murray, pp 190—198. Scriver, pp 1521—1552. Sack, pp 121-138. Wilson, pp 287-317.) The major fate of acetoacetyl CoA formed from condensation of acetyl CoA in the liver is the formation of 3-hydroxy-3-methylglutaryl CoA (HMG CoA). Under normal postabsorp-tive conditions, HMG CoA production occurs in the cytoplasm of hepatocytes as part of the overall process of cholesterol biosynthesis. However, in fasting or starving persons, as well as in patients with uncontrolled diabetes mellitus, HMG CoA production occurs in liver mitochondria as part of ketone body synthesis. In this process, HMG CoA is cleaved by HMG CoA lyase to yield acetoacetate and acetyl CoA. The NADH-dependent enzyme P-hydroxybutyrate dehydrogenase converts most of the acetoacetate to P-hydroxybutyrate, These two ketone bodies, acetoacetate and P-hydroxybutyrate, diffuse into the blood and are transported to peripheral tissues. 

Acetoacetate and (3-hydroxybutyrate can be oxidized as fuels in most tissues, including skeletal muscle, brain, certain cells of the kidney, and cells of the intestinal mucosa. Cells transport both acetoacetate and (3-hydroxybutyrate from the circulating blood into the cytosol, and into the mitochondrial matrix. Here p-hydrox-ybutyrate is oxidized back to acetoacetate by p-hydroxybutyrate dehydrogenase. This reaction produces NADH. Subsequent steps convert acetoacetate to acetyl CoA (Fig. 23.19). 

P-Hydroxybutyrate dehydrogenase (located in mitochondria) catalyses the conversion of acetoacetate to P-hydroxybutyrate. Acetone is formed by the spontaneous decarboxylation of acetoacetate (Fig. 1). Acetoacetate is also produced by degradation of the keto-plastic amino acids, leucine, isoleucine, phenylalanine and tyrosine. 

Acetoacetate is the primary metabolic product but a substantial proportion of it is reduced to /3-hydroxybutyrate under the influence of P-hydroxybutyrate dehydrogenase and NADH. Acetoacetate also undergoes a slow spontaneous decarboxylation to acetone which appears in the urine and, since it is volatile, is also lost from the lungs and may be smelt in the breath. 

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 ... 

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. 

Succinate, P-hydroxybutyric and glutamate dehydrogenase Catalase Caeruloplasmin Leucine aminopeptidase... 

The pH optimum for the lactate-to-pyruvate (L—>P) reaction is 8.8 to 9.8, and an assay mixture, optimized for LD-1 at 37 °C, contains NAD% 9mmol/L, and L-lactate, 80mmol/L. For the P —> L assay, at 37 °C, the pH optimum is 7.4 to 7.8, NADH 300fJ.mol/L, and pyruvate 0.85mmol/L. The optimal pH varies with the predominant isoenzymes in the sample and depends on the temperature and on substrate and buffer concentrations. The specificity of the enzyme extends from L-lactate to various related 2-hydroxyacids and 2-oxo-acids. The catalytic oxidation of 2-hydroxybutyrate, the next higher homologue of lactate, to 2-oxobutyrate is referred to as 2-hydroxybutyrate dehydrogenase (HBD) activity. LD does not act on n-lactate, and only NAD serves as a coenzyme. 

The penetrability of various thiol reagents through the inner mitochondrial membrane has been tested p-chloromercuribenzoate is a penetrant and inhibited d(—)-j8-hydroxybutyrate dehydrogenase in intact mitochondria on the inside of the mitochondrial membrane. 2-Chloromercuri-4-nitrophenol inhibited the ATPase of intact bovine heart mitochondria, and p-chloromercuribenzoate and mersalyl inhibited the liver microsomal calcium pump. While mersalyl had no effect on the Mg -dependent ATPase, adenylate cyclose from rat liver plasma was inhibited ... 

Somewhat surprisingly, within the mitochondria the ratio [NAD+]/[NADH] is 100 times lower than in the cytoplasm. Even though mitochondria are the site of oxidation of NADH to NAD+, the intense catabolic activity occurring in the P oxidation pathway and the citric acid cycle ensure extremely rapid production of NADH. Furthermore, the reduction state of NAD is apparently buffered by the low potential of the P-hydroxybutyrate-acetoacetate couple (Chapter 18, Section C,2). Mitochondrial pyridine nucleotides also appear to be at equilibrium with glutamate dehydrogenase. ... 

Fig. 23.18. Synthesis of the ketone bodies acetoacetate, P-hydroxybulyrate, and acetone. The portion of HMG-Co A shown in blue is released as acetyl CoA, and the remainder of the molecule forms acetoacetate. Acetoacetate is reduced to P-hydroxybutyrate or decarboxy-lated to acetone. Note that the dehydrogenase that interconverts acetoacetate and P-hydroxybutyrate is specific for the D-isomer. Thus, it differs from the dehydrogenases of P-oxidation, which act on 3-hydroxy acyl CoA derivatives and is specific for the L-isomer.

Nehlig, A., Crone, M.C. Lehr, P.R. (1980). Variations of 3-hydroxybutyrate dehydrogenase activity in brain and liver mitochondria of the developing chick. Biochim. Biophys. Acta, 633, 22-32. 

Polyhydroxyalkanoates biosynthesis is regulated, on one hand, by the activity of 3-ketothiolase (EC 2.3.1.16), and on the other hand of acetoacetyl-CoA reductase (EC 1.1.1.36) intracellular PHA breakdown is dependent on the activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30). Besides these three enzymes, the following compounds can be pointed out as major factors responsible of the activities of the key enzymes acetyl-CoA, free CoA, NAD(P) + (or NAD(P)H2, respectively) and, to a lower extent, ATP, pyruvate and oxalacetate. In any case, acetyl-CoA can be considered as the central metabolite both for biomass formation and PHB biosynthesis. This compound stems from the catabolic break down of carbon substrates like sugars (mainly catabolized by the 2-Keto-3-desoxy-6-phosphogluconate pathway) or fatty acids (converted by 6-oxidation). 

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. 

A complication arises if the p-dicarbonyl compound is substituted on the central carbon, since diastereomers could be formed. Nonetheless useful selectivity is often observed. The enzyme D-3-hydroxybutyrate dehydrogenase (HBDH) fromRhodopseudonwnas spheroides in the presence of NADH reduces only the (5)-enantiomer of 2-ketocyclohexanecarboxylic acid, whereas baker s yeast operates exclusively on the (/ )-antipode. 

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