Properties and substrate specificity of some reactions catalysed by a short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata

Abstract

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.

Biosynthesis of Saturated and Unsaturated Fatty Acids

“…A short-chain fatty acyl-CoA synthetase was described in extracts of ger minated seeds of Pinus radiata (Young and Anderson, 1974). The observed maximal velocity for propionate was 207 nmol/min/mg of protein, for acetate 166, for butyrate 135, and for valerate 25; the K m for propionate was 210 fiM, for butyrate 330 /JLM, for acetate 4700 yM, and for valerate 2100 fiM.…”

Spinach Leaf Acetyl-Coenzyme A Synthetase: Purification and Characterization

“…Millerd and Bonner (17) attributed the substrate nonspecificity to the presence of several activating enzymes. Young and Anderson (32) identified a short chain fatty acyl-CoA synthetase in extracts of germinated seeds of Pinus radiata which had a greater affinity for butyrate than for acetate, but no butyryl-CoA synthetase activity has been reported for leaf tissue. In contrast, acetylCoA synthetase purified from several different sources (4,7,10,29) activated acetate and propionate but not butyrate.…”

Analytical Methods for the Detection and Purification of ε-Poly-L-lysine for Studying Biopolymer Synthetases, and Bioelectroanalysis Methods for Its Functional Evaluation

“…32 However, most facilities are not equipped to handle radioactive materials. Thus, we have developed a colorimetric method for the determination of PPi (Fig.…”