The origin of chloroplastic acetyl coenzyme A

Pyruvic dehydrogenase activity has been examined in a number of highly purified leaf organelles. In spinach leaf cell, the major activity is in the mitochrondrion with low activity in isolated chloroplasts. The major source of CO₂ derived from pyruvic acid metabolism in the isolated chloroplast is via the acetolactic synthase reaction localized in the chloroplast. Evidence is presented that the leaf mitochondrion contains both the pyruvic acid dehydrogenase and an acetyl coenzyme A hydrolase. It is suggested that free acetic acid is generated in the mitochrondrion and then moves to the chloroplast where acetyl coenzyme synthetase converts it from the metabolically inert acid to the very metabolically active acetyl coenzyme A.     

A multisubunit acetyl coenzyme A carboxylase from soybean

A multisubunit form of acetyl coenzyme A (CoA) carboxylase (ACCase) from soybean (Glycine max) was characterized. The enzyme catalyzes the formation of malonyl CoA from acetyl CoA, a rate-limiting step in fatty acid biosynthesis. The four known components that constitute plastid ACCase are biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and the alpha- and beta-subunits of carboxyltransferase (alpha- and beta-CT). At least three different cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode alpha-CT. Whereas BC, BCCP, and alpha-CT are products of nuclear genes, the DNA that encodes soybean beta-CT is located in chloroplasts. Translation products from cDNAs for BC, BCCP, and alpha-CT were imported into isolated pea (Pisum sativum) chloroplasts and became integrated into ACCase. Edman microsequence analysis of the subunits after import permitted the identification of the amino-terminal sequence of the mature protein after removal of the transit sequences. Antibodies specific for each of the chloroplast ACCase subunits were generated against products from the cDNAs expressed in bacteria. The antibodies permitted components of ACCase to be followed during fractionation of the chloroplast stroma. Even in the presence of 0.5 M KCl, a complex that contained BC plus BCCP emerged from Sephacryl 400 with an apparent molecular mass greater than about 800 kD. A second complex, which contained alpha- and beta-CT, was also recovered from the column, and it had an apparent molecular mass of greater than about 600 kD. By mixing the two complexes together at appropriate ratios, ACCase enzymatic activity was restored. Even higher ACCase activities were recovered by mixing complexes from pea and soybean. The results demonstrate that the active form of ACCase can be reassembled and that it could form a high-molecular-mass complex.     

Substrate specificity of acetyl coenzyme A synthetase

Acetyl coenzyme A synthetase (EC 6.2.1.1) has been examined for its ability to accept various carboxylic acids as substrates in place of acetic acid. The activity of the enzyme with these substrates was monitored using a coupled enzyme assay and high pressure liquid chromatography (HPLC) analysis. Short chain carboxylic acids were found to be active including: propionic, acrylic, fluoroacetic, methacrylic, 3-chloropropionic, 3-bromopropionic, and propiolic. The kinetic parameters, Km and % Vmax of the carboxylic acid substrates, are reported and show that these acids are poorer substrates than acetic acid. Several of the acyl CoAs were synthesized on a preparative scale using enzyme catalysis, purified using preparative HPLC, and characterized using proton NMR spectroscopy. In the course of the NMR identification, a complete and fully resolved spectral assignment for all the protons of coenzyme A was made and is reported. The acyl-CoA analogs should be useful as substrate analogs and as potential affinity labels for enzymes that bind acetyl-CoA.     

Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis.

To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation.     

Regulation of acetyl coenzyme A synthetase in Escherichia coli

Cells of Escherichia coli growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. Here, we present evidence that this switch occurs primarily through the induction of acs and that the timing and magnitude of this induction depend, in part, on the direct action of the carbon regulator cyclic AMP receptor protein (CRP) and the oxygen regulator FNR. It also depends, probably indirectly, upon the glyoxylate shunt repressor IclR, its activator FadR, and many enzymes involved in acetate metabolism. On the basis of these results, we propose that cells induce acs, and thus their ability to assimilate acetate, in response to rising cyclic AMP levels, falling oxygen partial pressure, and the flux of carbon through acetate-associated pathways.     

Proteolysis of liver acetyl coenzyme A carboxylase by cathepsin B

Proteolysis of acetyl-CoA carboxylase was examined with cathepsin B. When chicken liver acetyl-CoA carboxylase was incubated with cathepsin B at pH 6.3, the native 220-kDa polypeptide was primarily cleaved into two polypeptides of 125 and 115 kDa, and further degraded to polypeptides of 100-50 kDa.