Acetyl coenzyme A and coenzyme A contents of growing Clostridium kluyveri as determined by isotope assays

CoASH and some of its acyl derivatives, especially acetyl-SCoA, occupy a central position in the energy metabolism of the anaerobic Clostridium kluyveri, both as intermediates and as regulatory effectors. The steady state concentrations of these compounds were determined in growing cultures of this organism using an anaerobic and fast deproteinization technique and radio isotope assays. Acetyl-SCoA was determined as [1-¹⁴C]citrate formed in the presence of [4-¹⁴C]oxaloacetate and citrate synthase; 0.49 mol/g cell wet wt. were found CoASH, CoAS-SCoA after borohydride reduction, and total acyl derivatives of coenzyme A after hydrolysis of the thiol esters were converted to thioethers with [2,3-¹⁴C]N-ethylmaleimide and brought to radiochemical purity by chromatographic methods. While disulfides of coenzyme A were undetectable, 0.13 mol CoASH and 1.17 mol of total acyl-SCoA per g wet wt. were found. These data are consistent with the regulatory scheme of the energy metabolism of C. kluyveri previously proposed.Abbreviations DTE dithioerythritol - NEM N-ethylmaleimide - NES N-ethylsuccinimide Enzymes (EC 2.7.2.1) Acetate kinase, ATP: acetate phosphotransferase - (EC 3.1.3.1) Alkaline phosphatase, orthophosphoric monoester phosphohydrolase - (GOT) Aspartate aminotransferase - (EC 2.6.1.1) L-aspartate:2-oxoglutarate aminotransferase - (CS) Citrate synthase - (EC 4.1.3.7) citrate oxaloacetate-lyase (pro 3S-CH₂COOacetyl-CoA) - (EC 2.8.3.8) CoA-transferase, acyl-CoA:acetate CoA-transferase - (EC 1.1.1.37) Malate dehydrogenase, L-malate:NAD⁺ oxidoreductase - (EC 1.18.1.3) NADH:ferredoxin reductase, ferredoxin:NAD⁺ oxidoreductase - (EC 3.1.4.1) Phosphodiesterase (snake venom), orthophosphoric diester phosphohydrolase - (EC 2.3.1.8) Phosphotransacetylase, acetyl-CoA:orthophosphate acetyltransferase - (EC 2.3.1.9) Thiolase, acetyl-CoA:acetyl-CoA C-acetyltransferase A preliminary account of this work has been given (Decker et al. 1976)     

Chemical events in chloropropionyl coenzyme A inactivation of acyl coenzyme A utilizing enzymes

Incubation of 3-chloropropionyl-CoA with 3-hydroxy-3-methylglutaryl-CoA synthase results in exchange of the C2 proton with solvent as inactivation of enzyme proceeds. This enzyme is also inhibited by S-acrylyl-N-acetylcysteamine; the limiting rate constant for inactivation by the acrylyl derivative (0.36 min-1) slightly exceeds the value measured for chloropropionyl-CoA (0.31 min-1). These observations support the intermediacy of acrylyl-CoA in the chloropropionyl-CoA-dependent inactivation of hydroxymethylglutaryl-CoA synthase. Inhibition of fatty acid synthase by chloropropionyl-CoA is primarily due to alkylation of a reactive cysteine, although secondary reaction with the enzyme's pantetheinyl sulfhydryl occurs. Modification of fatty acid synthase by S-acrylyl-N-acetylcysteamine occurs at a limiting rate (1.8 min-1) that is comparable to that estimated for chloropropionyl-CoA-dependent inactivation. However, this enzyme lacks the ability to deprotonate C2 of an acyl group such as the chloropropionyl moiety. Since such a step would be required to generate an acrylyl group from chloropropionyl-S-enzyme, it is likely that a typical affinity labeling process accounts for inactivation of fatty acid synthase by chloropropionyl-CoA. HMG-CoA lyase is also inhibited by S-acrylyl-N-acetylcysteamine. In contrast to the ability of this reagent to serve as a mechanism-based inhibitor of hydroxymethylglutaryl-CoA synthase and an affinity label of fatty acid synthase, it acts as a group-specific reagent in modifying HMG-CoA lyase (kappa 2 = 86.7 M-1 min-1).     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K_m values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ～85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ～64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A: diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K(m) values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9+/-2.1 microM and 13.9+/-0.3 microM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits approximately 85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl-oleate formation without influencing the retinyl-palmitate formation. Using this inhibitor, we estimate that approximately 64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

Feedback regulation of murine pantothenate kinase 3 by coenzyme A and coenzyme A thioesters

Pantothenate kinase catalyzes a key regulatory step in coenzyme A biosynthesis, and there are four mammalian genes that encode isoforms of this enzyme. Pantothenate kinase isoform PanK3 is highly related to the previously characterized PanK1beta isoform (79% identical, 91% similar), and these two almost identical proteins are expressed most highly in the same tissues. PanK1beta and PanK3 had very similar molecular sizes, oligomeric form, cytoplasmic cellular location, and kinetic constants for ATP and pantothenate. However, these two PanK isoforms possessed distinct regulatory properties. PanK3 was significantly more sensitive to feedback regulation by acetyl-CoA (IC50 = 1 microm) than PanK1beta (IC50 = 10 microm), and PanK3 was stringently regulated by long-chain acyl-CoA (IC50 = 2 microm), whereas PanK1beta was not. Domain swapping experiments localized the difference in the two proteins to a 48-amino-acid domain, where they are the most divergent. Consistent with these more stringent regulatory properties, metabolic labeling experiments showed that coenzyme A (CoA) levels in cells overexpressing PanK3 were lower than in cells overexpressing an equivalent amount of PanK1beta. Thus, the distinct regulatory properties exhibited by the family of the pantothenate kinases allowed the rate of CoA biosynthesis to be controlled by regulatory signals from CoA thioesters involved in different branches of intermediary metabolism.