Properties and substrate specificity of some reactions catalysed by a short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata.
1. Crude extracts of seeds of Pinus radiata catalysed acetate-, propionate-, n-butyrate- and n-valerate-dependent PP(i)-ATP exchange in the presence of MgCl(2), which was apparently due to a single enzyme. Propionate was the preferred substrate. Crude extracts did not catalyse medium-chain or long-chain fatty acid-dependent exchange. 2. Ungerminated dry seeds contained short-chain fatty acyl-CoA synthetase activity. The activity per seed was approximately constant for 11 days after imbibition and then declined. The enzyme was located only in the female gametophyte tissue. 3. The synthetase was purified 70-fold. 4. Some properties of the enzyme were studied by [(32)P]PP(i)-ATP exchange. K(m) values for acetate, propionate, n-butyrate and n-valerate were 4.7, 0.21, 0.33 and 2.1mm respectively. Competition experiments between acetate and propionate demonstrated that only one enzyme was involved and confirmed that the affinity of the enzyme for propionate was greater than that for acetate. CoA inhibited fatty acid-dependent PP(i)-ATP exchange. The enzyme catalysed fatty acid-dependent [(32)P]PP(i)-dATP exchange. 5. The enzyme also catalysed the fatty acyl-AMP-dependent synthesis of [(32)P]ATP from [(32)P]PP(i). Apparent K(m) (acetyl-AMP) and apparent K(m) (propionyl-AMP) were 57mum and 7.5mum respectively. The reaction was inhibited by AMP and CoA. 6. Purified enzyme catalysed the synthesis of acetyl-CoA and propionyl-CoA. Apparent K(m) (acetate) and apparent K(m) (propionate) were 16mm and 7.5mm respectively. The rate of formation of acetyl-CoA was enhanced by pyrophosphatase. 7. It was concluded that fatty acyl adenylates are intermediates in the formation of the corresponding fatty acyl-CoA.
D000085:Acetates / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D002087:Butyrates / Q000378:metabolism; D002250:Carbon Radioisotopes; D002384:Catalysis; D002498:Centrifugation; D002712:Chlorides / Q000494:pharmacology; D002850:Chromatography, Gel; D003066:Coenzyme A Ligases / Q000302:isolation & purification / Q000378:metabolism*; D011756:Diphosphates / Q000378:metabolism; D004355:Drug Stability; D005227:Fatty Acids / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008968:Molecular Conformation; D010761:Phosphorus Radioisotopes; D011422:Propionates / Q000494:pharmacology; D011755:Pyrophosphatases; D012639:Seeds / Q000201:enzymology*; D014197:Trees; D014631:Valerates / Q000378:metabolism
D000085:Acetates; D002087:Butyrates; D002250:Carbon Radioisotopes; D002712:Chlorides; D011756:Diphosphates; D005227:Fatty Acids; D010761:Phosphorus Radioisotopes; D011422:Propionates; D014631:Valerates; D000255:Adenosine Triphosphate; D011755:Pyrophosphatases; D003066:Coenzyme A Ligases; D008274:Magnesium