Pyruvate carboxylases and

COMPARTMENTALIZED PYRUVATE CARBOXYLASE DEPENDS ON METABOLITE CONVERSION AND TRANSPORT The second interesting feature of pyruvate carboxylase is that it is found only in the matrix of the mitochondria. By contrast, the next enzyme in the gluconeogenic pathway, PEP carboxykinase, may be localized in the cytosol or in the mitochondria or both. For example, rabbit liver PEP carboxykinase is predominantly mitochondrial, whereas the rat liver enzyme is strictly cytosolic. In human liver, PEP carboxykinase is found both in the cytosol and in the mitochondria. Pyruvate is transported into the mitochondrial matrix, where it can be converted to acetyl-CoA (for use in the TCA cycle) and then to citrate (for fatty acid synthesis see Figure 25.1). /Uternatively, it may be converted directly to 0/ A by pyruvate carboxylase and used in glu-... 

Step 1 of Figure 29.13 Carboxylation Gluconeogenesis begins with the carboxyl-afion of pyruvate to yield oxaloacetate. The reaction is catalyzed by pyruvate carboxylase and requires ATP, bicarbonate ion, and the coenzyme biotin, which acts as a carrier to transport CO2 to the enzyme active site. The mechanism is analogous to that of step 3 in fatty-acid biosynthesis (Figure 29.6), in which acetyl CoA is carboxylated to yield malonyl CoA. 

Rose, I. A. Stereochemistry of pyruvate kinase, pyruvate carboxylase, and malate enzyme reactions. J. Biol. Chem. 245, 6052—6056 (1970). 

Carbonic anhydrase (CA, also called carbonate dehydratase) is an enzyme found in most human tissues. As well as its renal role in regulating pH homeostasis (described below) CA is required in other tissues to generate bicarbonate needed as a co-substrate for carboxylase enzymes, for example pyruvate carboxylase and acetyl-CoA carboxylase, and some synthase enzymes such as carbamoyl phosphate synthases I and II. At least 12 isoenzymes of CA (CA I—XII) have been identified with molecular masses varying between 29 000 and 58 000 some isoenzymes are found free in the cytosol, others are membrane-bound and two are mitochondrial. 

Coordinate Regulation of Pyruvate Carboxylase and Pyruvate Dehydrogenase by Acetyl CoA... 

The two major mitochondrial enzymes (Figure 1-14-5) that use pyruvate, pyruvate carboxylase and pyruvate dehydrogenase, are both regulated by acetyl CoA. This control is important in these contexts ... 

Between meals when fatty acids are oxidized in the liver for energy, accumulating acetyl CoA activates pyruvate carboxylase and gluconeogenesis and inhibits PDH, thus preventing conversion of lactate and alanine to acetyl CoA. 

Answer B. Acetyl CoA activates pyruvate carboxylase and gluconeogenesis during fasting. 

The pyrnvate/phosphoenolpyrnvate cycle, which involves the enzymes pyrnvate kinase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase. 

Fung, C. H., Mildvan, A. S., Allerhand, A, Komoroski, R., and Scrutton, M. C. (1973). Interaction of pyruvate with pyruvate carboxylase and pyruvate kinase as studied by paramagnetic effects on relaxation rates. Biochemistry 12, 620-629. 

Diabetes - insulin dependent Methyl malonic, propionic or isovaleric acidaemias Pyruvate carboxylase and multiple carboxylase deficiency Gluconeogenesis enzyme deficiency glucose-6-phosphatase, fructose-1,6-diphosphatase or abnormality of glycogen synthesis (glycogen synthase) Ketolysis defects Succinyl coenzyme A 3-keto acid transferase ACAC coenzyme A thiolase... 

Robinson, B.H., MacKay, N., Chun, K., Ling, M. (1996) Disorders of pyruvate carboxylase and the pyruvate dehydrogenase complex. J. Inherit. Metab. Dis. 19, 452-462. 

Pyruvate is converted to phosphoenolpyruvate (PEP) by pyruvate carboxylase and PEP carboxykinase. The carboxylase requires biotin and ATP, and is allosterically activated by acetyl CoA. PEP carboxykinase, which requires GTP, is the rate-limiting step in gluconeo genesis. The transcription of its mRNA is increased by glucagon and decreased by insulin. 

When the carbohydrates are being metabolized, TCA ycle intermediates are replenished by production of oxalo-etate from pyruvate. In mammals, this reaction is cata-[zed by pyruvate carboxylase, and one ATP-to-ADP concision is associated with the carboxylation. Other operties of this reaction are discussed later in this chapter l connection with regulation of the TCA cycle and related [etabolic sequences. 

ADP, acetyl-CoA, succinyl-CoA, and citrate. The major known sites for regulation of the cycle include two enzymes outside the cycle (pyruvate dehydrogenase and pyruvate carboxylase) and three enzymes inside the cycle (citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase). All of these sites of regulation represent important metabolic branchpoints. 

Gluconeogenesis uses seven of the reactions in glycolysis, but three are replaced by the sum of the pyruvate carboxylase and phosphoeno/pyruvate carboxykinase reactions, the fructose 1,6-biphosphatase reaction, and the glucose 6-phosphatase reaction. Tables 4.7, 4.8, and 4.9 give the thermodynamic properties of these reactions and the net reaction for gluconeogenesis. 

Weber LW, Lebofsky M, Stahl BU, et al. 1992a. Comparative toxicity of four chlorinated dibenzo-p-dioxins (CDDs) and their mixture. Part II Structure-activity relationships with inhibition of hepatic phospholpyruvate carboxykinase, pyruvate carboxylase, and gamma-glutamyl transpeptidase activities. Arch Toxicol 66(7) 478-483. 

Fig. 1. Comparison of gluconeogenesis and glycolysis. The three steps of glycolysis that are irreversible are numbered. (1) Flexokinase in glycolysis is reversed by glucose 6-phosphatase in gluconeogenesis (2) PFK in glycolysis is reversed by fructose 1,6-bisphosphatase in gluconeogenesis (3) pyruvate kinase in glycolysis is reversed by two sequential reactions in gluconeogenesis catalyzed by pyruvate carboxylase and PEP carboxykinase.

Thus, reversal of the glycolytic step from PEP to pyruvate requires two reactions in gluconeogenesis, pyruvate to oxaloacetate by pyruvate carboxylase and oxaloacetate to PEP by PEP carboxykinase. Given that the conversion of PEP to pyruvate in glycolysis synthesizes ATP, it is not surprising that the overall reversal of this step needs the input of a substantial amount of energy, one ATP for the pyruvate carboxylase step and one GTP for the PEP carboxy kinase step. 

Regulation of pyruvate kinase, pyruvate carboxylase and PEP carboxykinase... 

There have been reports of other glucagon actions in the liver which can be related to the elevation of cAMP, but whose molecular mechanisms are not well defined. Examples are the stimulations of ketogenesis, ureogenesis, amino acid transport, respiration and ion fluxes, the rapid changes in pyruvate dehydrogenase and pyruvate carboxylase, and the induction of P-enolpyruvate carboxykinase and other enzymes. 

Robinson BH Lactic academia disorders of pyruvate carboxylase and pyruvate dehydrogenase, in Scriver CR, Beaudet AL, Sly WS, Valle D (eds), The Metabolic and Molecular Bases of Inherited Disease. 8th ed. McGraw-Hill, New York, 2001, pp. 2275-2296. 

In addition, hepatic fatty acid oxidation is also required to sustain gluconeogenesis. These fatty acids may be obtained from exogenous feeding or metabolism of fatty acids released from endogenous lipid stores. (3-Oxidation of fatty acids provides the acetyl-CoA needed to activate mitochondrial pyruvate carboxylase and the NADH used as the substrate in the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase in the direction of gluconeogenesis (see Fig. 10-1). 

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