Carboxylase acetivity

Rittenberg and Bloch showed in the late 1940s that acetate units are the building blocks of fatty acids. Their work, together with the discovery by Salih Wakil that bicarbonate is required for fatty acid biosynthesis, eventually made clear that this pathway involves synthesis of malonyl-CoA. The carboxylation of acetyl-CoA to form malonyl-CoA is essentially irreversible and is the committed step in the synthesis of fatty acids (Figure 25.2). The reaction is catalyzed by acetyl-CoA carboxylase, which contains a biotin prosthetic group. This carboxylase is the only enzyme of fatty acid synthesis in animals that is not part of the multienzyme complex called fatty acid synthase. 

The biotin-dependent carboxylases that couple the decarboxylation of malonate to acetate in Malonomonas rubra to the transport of Na across the cytoplasmic membrane... 

ACETATE KINASE ACETYL-CoA CARBOXYLASE ACETYL-CoA SYNTHETASE N-ACETYLCLUCOSAMINE KINASE ACTIN ATPase ACTOMYOSIN ATPase N-ACYLMANNOSAMINE KINASE ADENINE NUCLEOTIDE TRANSLOCASE ADENOSINE KINASE ADENYLATE KINASE (MYOKINASE) ADENYLYLSULFATE KINASE d-ALANINE-d-ALANINE LIGASE... 

Orthophosphate as substrate or product, ACETATE KINASE (PYROPHOSPHATE) ACETYL-CoA CARBOXYLASE ACID PHOSPHATASE ACTOMYOSIN ATPase ACYL PHOSPHATASE ASPARTATE-SEMIALDEHYDE DEHYDROGENASE ATPases... 

In the ruminant mammary tissue, it appears that acetate and /3-hydroxybutyrate contribute almost equally as primers for fatty acid synthesis (Palmquist et al. 1969 Smith and McCarthy 1969 Luick and Kameoka 1966). In nonruminant mammary tissue there is a preference for butyryl-CoA over acetyl-CoA as a primer. This preference increases with the length of the fatty acid being synthesized (Lin and Kumar 1972 Smith and Abraham 1971). The primary source of carbons for elongation is malonyl-CoA synthesized from acetate. The acetate is derived from blood acetate or from catabolism of glucose and is activated to acetyl-CoA by the action of acetyl-CoA synthetase and then converted to malonyl-CoA via the action of acetyl-CoA carboxylase (Moore and Christie, 1978). Acetyl-CoA carboxylase requires biotin to function. While this pathway is the primary source of carbons for synthesis of fatty acids, there also appears to be a nonbiotin pathway for synthesis of fatty acids C4, C6, and C8 in ruminant mammary-tissue (Kumar et al. 1965 McCarthy and Smith 1972). This nonmalonyl pathway for short chain fatty acid synthesis may be a reversal of the /3-oxidation pathway (Lin and Kumar 1972). 

Both methylmalonic aciduria and propionyl-CoA decarboxylase deficiency are usually accompanied by severe ketosis, hypoglycemia, and hyperglycinemia. The cause of these conditions is not entirely clear. However, methylmalonyl-CoA, which accumulates in methylmalonic aciduria, is a known inhibitor of pyruvate carboxylase. Therefore, ketosis may develop because of impaired conversion of pyruvate to oxalo-acetate. 

The rate limiting step in fatty acid synthesis is catalyzed by acetyl-CoA carboxylase to produce malonyl-CoA at the expense of one ATP.31 Malonate and acetate are transferred from CoA to acyl carrier protein in the cytosolic fatty acid synthetase complex, where chain extension leads to the production of palmitate. Palmitate can then be transferred back to CoA, and the chain can be extended two carbons at a time through the action of a fatty acid elongase system located in the endoplasmic reticulum. The >-hydroxylation that produces the >-hydroxyacids of the acylceramides is thought to be mediated by a cytochrome p450 just when the fatty acid is long enough to span the endoplasmic reticular membrane. 

OAA by pyruvate carboxylase (EC 6.4.1.1), thereby completing the net transport of the C2 unit (acetate) from the mitochondrion to the cytosol with the added advantage of having converted a reducing equivalent as NADH + H+ to NADPH + H+. This mechanism of C2 transport provides up to 50% of the NADPH + H+ for fatty acid synthesis in nonruminants. 

The first committed step for the incorporation of acetate carbon into fatty acids is mediated by acetyl-CoA carboxylase (ACC EC 6.4.1.2) in two steps, as follows (Allred and Reilly, 1997) ... 

The pathway from acetate to palmitic acid (actually a palmitic acid-acyl carrier protein complex) involves at least nine enzymes acetyl CoA synthetase, acetyl CoA carboxylase, and the seven enzyme fatty acid synthetase complex. We chose first to test the effect of these compounds on acetyl CoA carboxylase (ACCase) activity. There were several reasons to select ACCase as the... 

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). 

Is a specific Inhibitor of type II fatty acid synthetase In higher plants and . coll 12401. The acetyl-CoA ACP S-acety1-transferase Is the apparent specific site of Inhibition 12411. Another antibiotic, cerulenin (structure not shown) Inhbits -ketococy1-ACP synthetase I In bacteria, fungi, and plants, but also Is Inhibitory to other sites such as polyketide and sterol biosynthesis 1242-2441. Cerulenin and thiolactomycin Inhibited CQ14W-acetate Incorporation Into fatty acids at 150 values of 50 and 4 uM, respectively 12451. Recently cydohexanedlone herbicides have been shown to Inhibit lipid biosynthesis by Inhibition of acetyl-CoA carboxylase 12461. 

Pyruvate carboxylase also requires a monovalent cation for activity. The activity of the purified enzyme was measured in the presence of various monovalent cations, as indicated in Table 10.7. Similar patterns of stimulation have been fovmd for acetyl-CoA synthetase, an enzyme used in acetate metabolism (Webster, 1966), propionyl-CoA carboxylase (Giorgio and Plaut, 1967), and several other enzymes (Suelter, 1970). Maximal activity of the aforementioned enzymes usually occurs at a wide range of potassium concentrations, that is, from 50 to 150 mM. There is therefore little reason to believe that the slight changes in intracellular K concentrations that can occur under normal conditions or during K deficiency result in an impairment in the activities of these enzymes or in some t)q>e of regulation of the activities. 

Activity-levels of phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49, PEPK) were examined with Rhodopseudomonas sp. No.7 grown photoanaerobically in an ethanol-bicarbonate and in an acetate medium. PEPC and PEPK were purified from cells grown under these conditions, and several characteristics of the enzymes were discussed in connection with photoheterotrophy of purple nonsulfur bacteria. 

In a typical experiment, 20 mg of thermal polymer in 20 ml of autoclaved 0.2 N tris buffer, pH 8.3, was incubated at 37.5° with 2.0 pCi of sodium l-i C-p3Tuvate (specific activity, 5/iCi//xmole, giving a substrate/polymer ratio of 0.02 /.imoles/mg). Incubation was usually for 24 hours, but occasionally for 48 or 72 hours, after which the liberated COa was assayed as barium carbonate. Similar results were obtained when radioactive acetate was counted. Aseptic precautions were taken during the preparation and assay of the polymers. Activities (cpm per 20 mg polymer per 24 hours. Table IX) were 30,000-33,000 for acidic 2 2 1-proteinoids, 33,000-41,000 for 1 1 1-proteinoids, and 33,000 for 1 1 3-proteinoid. A lysine-rich proteinoid gave a value of 15,800. Hydrolysates of polymers and unpolymerized mixtures of amino acids had activities of 4000-5000, only slightly greater than that of the spontaneous control (1000-3000). Enzymes other than carboxylase and other proteins gave values of 2000-5000. 

Even if the incorporation of [ C]acetate into lipids has been observed (M3), a de novo synthesis of fatty acid is not possible due to the lack of acetyl-CoA carboxylase (EC 6.4.1.2) (Ml). The incorporation of acetate into fatty acids represents chain elongation of preformed fatty acids. 

CH..CO.COOH + (carboxylase enzyme) = CH.CHO 4- CO pyruvic acid acetic aldehyde... 

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