Reengineering the specificity of a serine active-site enzyme. Two active-site mutations convert a hydrolase to a transferase.

Two residues are known to play important catalytic roles in fatty acyl-thioester hydrolase, thioesterase II: Ser-101, the site of a covalent acyl-enzyme intermediate, and His-237 which is within hydrogen bonding distance of Ser-101 and facilitates catalysis by increasing the nucleophilicity of this residue. In this study we have examined the effect of mutations at these two residues on the ability of the enzyme to function as a hydrolase and, in the presence of a thiol acceptor, as an acyltransferase. In the hydrolase reaction kcat values for the wild-type, H237R, S101C, and S101C, H237R thioesterase enzymes were 0.11, < 0.002, 0.10, and < 0.002 s-1, respectively, and at steady state, the proportion of each enzyme present as the covalent acyl-enzyme intermediate was 11, 91, 71, and 100%, respectively. In the acyltransferase reaction no activity could be detected for the wild-type or H237R enzymes but the specific activities of the S101C and S101C/H237R thioesterases were 170 and 1300 nmol/min/mg of protein, respectively. From this data we conclude the following: the wild-type enzyme functions exclusively as a hydrolase. The H237R mutant acts ineffectively as a hydrolase primarily because the deacylation reaction is drastically retarded. The S101C enzyme functions well as a hydrolase, even though the rate of deacylation is adversely affected, and this enzyme can also perform as an acyltransferase. Mutation of both catalytic residues leads to a complete loss of hydrolase activity and the S101C,H237R mutant functions as an effective acyltransferase exhibiting kcat values higher then those of the wild-type enzyme acting as a hydrolase. This study reveals that, with only two amino acid replacements, an enzyme capable of functioning exclusively as a hydrolase can be converted into an equally active enzyme performing solely as an acyltransferase.