Studies on the Mechanism of Fatty Acid Synthesis: XX. Preparation and General Properties of β-Hydroxybutyryl Acyl Carrier Protein Dehydrase

Abstract

Abstract An enzyme, β-hydroxybutyryl acyl carrier protein (ACP) dehydrase, which catalyzes the reversible dehydration of short chain β-hydroxyacyl-ACP to the corresponding α, β-unsaturated acyl-ACP derivatives has been isolated from extracts of Escherichia coli. Dehydrase preparations of specific activity of 5000 mµmoles per min per mg of protein were homogeneous as assessed by ultracentrifugal analysis and by polyacrylamide disc gel electrophoresis. The enzyme has an estimated molecular weight of 26,000. It is relatively heat-stable and is active over a broad pH range with maximal activity between pH 7.5 and 8.5. The dehydrase has a functional —SH group and can be readily inhibited by —SH-binding reagents such as N-ethylmaleimide and iodoacetamide. The dehydrase reaction is readily reversible and the equilibrium constant for the dehydration of β-hydroxybutyryl-ACP is estimated to be 19 m. The β-hydroxybutyryl-ACP dehydrase is active on ACP thioesters but inactive on the coenzyme A derivatives. The enzyme is specific for short chain length β-hydroxyacyl-ACP derivatives (C4 to C8). The estimated maximal rates of hydration of crotonyl-ACP, 2-hexenoyl-ACP, and 2-octenoyl-ACP are 4100, 2300, and 200 mµmoles per min per mg, respectively. The enzyme is inactive on 2-decenoyl-ACP. This limited chain length specificity of the β-hydroxybutyryl-ACP dehydrase is responsible for the accumulation of β-hydroxydecanoyl-ACP in a reconstituted fatty acid-synthesizing system comprised of highly purified preparations of malonyl-CoA-ACP transacylase, acyl-malonyl-ACP condensing enzyme, β-ketoacyl-ACP reductase, β-hydroxybutyryl-ACP dehydrase, and enoyl-ACP reductase. The β-hydroxy-decanoyl-ACP thus synthesized is readily converted to either saturated or unsaturated fatty acids by the action of protein fractions which contain at least two long chain βhydroxyacyl-ACP dehydrases.