Hydroxybutyrate dehydrogenase reaction

Once equilibrium is established for the 3-hydroxybutyrate dehydrogenase reaction, the following equation applies ... 

Ketogenesis required three specific reactions which have nothing directly to do with fatty acid degradation (Krebs t aJL., 1971). These reactions are the HMG-CoA synthase reaction, the HMG-CoA lyase reaction, and the 3-hydroxybutyrate dehydrogenase reaction ... 

Ketone body synthesis occurs only in the mitochondrial matrix. The reactions responsible for the formation of ketone bodies are shown in Figure 24.28. The first reaction—the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA—is catalyzed by thiolase, which is also known as acetoacetyl-CoA thiolase or acetyl-CoA acetyltransferase. This is the same enzyme that carries out the thiolase reaction in /3-oxidation, but here it runs in reverse. The second reaction adds another molecule of acetyl-CoA to give (i-hydroxy-(i-methyl-glutaryl-CoA, commonly abbreviated HMG-CoA. These two mitochondrial matrix reactions are analogous to the first two steps in cholesterol biosynthesis, a cytosolic process, as we shall see in Chapter 25. HMG-CoA is converted to acetoacetate and acetyl-CoA by the action of HMG-CoA lyase in a mixed aldol-Claisen ester cleavage reaction. This reaction is mechanistically similar to the reverse of the citrate synthase reaction in the TCA cycle. A membrane-bound enzyme, /3-hydroxybutyrate dehydrogenase, then can reduce acetoacetate to /3-hydroxybutyrate. 

Hydroxymethylglutaryl-CoA lyase 4.1.3.4 3-Hydroxybutyrate dehydrogenase 1.1.1.30 Nonenzymatic reaction... 

Phospholipid vesicles (and bilayers) composed of phospholipids with well-defined fatty acid side chains undergo a sharp transition from a crystallinelike state to an amorphous state as the temperature is raised.107 The transition temperature depends on the nature of the fatty acid side chains. For example, for C12 saturated fatty acid chains on lecithin the transition temperature is 0° and for C18 saturated fatty acid chains it is 58°C for unsaturated lecithins the transition temperature is below zero.107 For real membranes sharp phase transitions are not observed, because of the heterogeneous composition of the membrane. In the case of /3 hydroxybutyrate dehydrogenase, the enzymic activity apparently is not influenced by this phase transition as judged by the temperature dependence of the reaction rate. However, for some membrane-bound proteins, a plot of the reaction rate versus the reciprocal temperature... 

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. 

Figure 22.19. Formation of Ketone Bodies. The Ketone bodies-acetoacetate, d-3-hydroxybutyrate, and acetone from acetyl CoA are formed primarily in the liver. Enzymes catalyzing these reactions are (1) 3-ketothiolase, (2) hydroxymethylglutaryl CoA synthase, (3) hydroxymethylglutaryl CoA cleavage enzyme, and (4) d-3-hydroxybutyrate dehydrogenase. Acetoacetate spontaneously decarboxylates to form acetone.

Acetoacetate may be reduced by an NAD-requiring dehydrogenase (3-hydroxybutyrate dehydrogenase) to 3-hydroxybutyrate. This is a reversible reaction. 

Acetoacetate may enter cells directly, or it may be produced from the oxidation of 3-hydroxybutyrate by 3-hydroxybutyrate dehydrogenase. NADH is produced by this reaction and can generate ATP. 

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. 

Acetoacetate is a ketone body that participates in numerous metabolic reactions. Enzymes acting on it include HMG-CoA lyase, / -hydroxybutyrate dehydrogenase, and acetoacetate decarboxylase... 

Acetoacetate can directly enter the blood or it can be reduced by (3-hydroxybu-tyrate dehydrogenase to (3-hydroxybutyrate, which enters the blood (see Fig. 23.18). This dehydrogenase reaction is readily reversible and interconverts these two ketone bodies, which exist in an equilibrium ratio determined by the NADH/NAD ratio of the mitochondrial matrix. Under normal conditions, the ratio of (3-hydroxybutyrate to acetoacetate in the blood is approximately 1 1. 

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). 

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. 

The enzyme hydroxyacyl dehydrogenase described above is specific for the L-isomer. Apparently, some mammalian tissue can also oxidize the D-isomer, but it is not clear what enzymic mechanism is responsible for that reaction. Although the presence of an enzyme that specifically catalyzes the oxidation of the D-hy-droxyacyl ester to yield the keto acid has been proposed by some, others believe that the D-hydroxyacyl is transformed to the L-hydroxy acid by enzymes with racemase activity—namely, crotonase and another racemase. The equilibrium of the enzyme reaction is modified by the presence of magnesium in the medium. The modification of the equilibrium probably results from the complexion of magnesium with the keto acid. Eliminating the product favors the formation of the hydroxyacyl. Hydroxybutyrate can also be oxidized by an enzyme found in the mitochondria of many tissues, such as brain, kidney, heart, and liver. Hydroxybutyrate dehydrogenase has been isolated, solubilized, purified from beef heart, and demonstrated to require lecithin for activity. 

Acetoacetate Metabolism. An active deacylase in liver is responsible for the formation of free acetoacetate from its CoA derivative. The j8-hydroxybutyric dehydrogenase mentioned above and a decarboxylase are capable of converting acetoacetate into the other ketone bodies, /3-hydroxybutyrate, and acetone. liver does not contain a mechanism for activating acetoacetate. Heart muscle has been found to contain a specific thiophorase that forms acetoacetyl CoA at the expense of suc-cinyl CoA. Acetoacetate is thus used by peripheral tissues by activation through transfer, then reaction with either the enzymes of fatty acid synthesis or jS-ketothiolase and the enzymes that use acetyl CoA. 

Figure 3 Synthesis of ketone bodies. In the mitochondria of hepatocytes, acetyl-CoA derived from /3-oxidation is converted to ketone bodies, primarily acetoacetate and /3-hydroxybutyrate, rather than entering the tricarboxylic acid cycle. Two molecules of acetyl-CoA condense in a reversal of the last /3-oxidation reaction (3-oxoacyl-CoA thiolase). The product, acetoacetyl-Ck)A, condenses with another molecule of acetyl-CoA, yielding /3-hydroxy, /3-methyl-glutaryl-CoA (HMG-CoA), a reaction catalysed by HMG-CoA synthase. Cleavage of HMG-CoA by HMG-CoA lyase yields acetoacetate, regenerating one molecule of acetyl-CoA. Acetoacetate is reversibly reduced to /3-hydroxybutyrate via the NAD-dependent enzyme /3-hydroxybutyrate dehydrogenase. These ketone bodies can traverse the inner mitochondrial membrane, eventually reaching the bloodstream for ultimate use by the brain and other tissues.

---