The sulfur atoms of the substrate CoA and the catalytic cysteine are required for a productive mode of substrate binding in bacterial biosynthetic thiolase, a thioester-dependent enzyme.

Thioesters are more reactive than oxoesters, and thioester chemistry is important for the reaction mechanisms of many enzymes, including the members of the thiolase superfamily, which play roles in both degradative and biosynthetic pathways. In the reaction mechanism of the biosynthetic thiolase, the thioester moieties of acetyl-CoA and the acetylated catalytic cysteine react with each other, forming the product acetoacetyl-CoA. Although a number of studies have been carried out to elucidate the thiolase reaction mechanism at the atomic level, relatively little is known about the factors determining the affinity of thiolases towards their substrates. We have carried out crystallographic studies on the biosynthetic thiolase from Zoogloea ramigera complexed with CoA and three of its synthetic analogues to compare the binding modes of these related compounds. The results show that both the CoA terminal SH group and the side chain SH group of the catalytic Cys89 are crucial for the correct positioning of substrate in the thiolase catalytic pocket. Furthermore, calorimetric assays indicate that the mutation of Cys89 into an alanine significantly decreases the affinity of thiolase towards CoA. Thus, although the sulfur atom of the thioester moiety is important for the reaction mechanism of thioester-dependent enzymes, its specific properties can also affect the affinity and competent mode of binding of the thioester substrates to these enzymes.