β-Hydroxydecanoyl Thioester Dehydrase

Abstract

Abstract β-Hydroxydecanoyl thioester dehydrase from E. coli has been shown to have a molecular weight of 36,000 by ultracentrifugation, gel filtration, and amino acid composition. Electrophoretic behavior, however, suggests that the protein molecule is composed of two equal, if not identical, polypeptide chains of molecular weight 18,000. The enzyme also contains 4 half-cystinyl residues per 36,000 g of protein. A variety of chemical modifications, including photooxidation, alkylation, and nitration, lead to the conclusion that 1 histidyl and 1 tyrosyl residue are essential for the catalytic activity of the enzyme. The pH-kcat profile for the isomerization reaction is consistent with the participation of these two amino acids in the catalytic process. Evidence is presented that 3-decynoyl-N-acetylcysteamine, a specific dehydrase inhibitor, reacts covalently with the same histidyl residue and protects the enzyme against the modifying active site-directed reagents. Conversely, nitrated or alkylated enzyme has lost the ability to react with the acetylenic inhibitor. No treatment has been found which will dissociate dehydration from isomerization activity, supporting the concept that the dehydrase is a truly multifunctional enzyme.