Conversion of serine-114 to cysteine-114 and the role of the active site nucleophile in acyl transfer by myristoyl-ACP thioesterase from Vibrio harveyi.

The lux-specific myristoyl-ACP thioesterase (LuxD) is responsible for diverting myristic acid into the luminescent system and can function as an esterase and transferase as well as cleave myristoyl-CoA and other thioesters. The recently elucidated crystal structure of the enzyme shows that it belongs to the alpha/beta hydrolase family and that it contains a typical catalytic triad composed of Asp211, His241, and Ser114. What is unusual is that the nucleophilic S114 is not contained within the esterase consensus motif GXSXG although the stereochemistry of the turn involving S114 is almost identical to the nucleophilic elbow found in alpha/beta hydrolases. In contrast to mammalian thioesterases, deacylation of LuxD was the rate-limiting step, with the level of acylated enzyme formed on reaction with myristoyl-CoA and the pre-steady-state burst of p-nitrophenol on cleavage of p-nitrophenyl myristate both being 0.7 mol/mol. Cold chase experiments showed that the deacylation rate of LuxD corresponded closely to the turnover rate of the enzyme with ester or thioester substrates. Replacement of S114 by a cysteine residue generated a mutant (S114C) that was acylated with the same pH dependence as LuxD but had greatly diminished capacity to transfer acyl groups to water or glycerol. The acyl group could be removed from the S114C mutant by neutral hydroxylamine, showing that a cysteine residue had been acylated. Mutation of H241 creating the double mutant, S114C.H241N, decreased acylation of the cysteine residue. These results provide direct kinetic and chemical evidence that S114 is the site of acylation linked to H241 in the charge relay system and have led to the recognition of a more general consensus motif flanking the nucleophilic serine in thioesterases.