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 a s an esterase and transferase as well as cleave myristoyl-CoA and othe r thioesters, The recently elucidated crystal structure of the enzyme shows that it belongs to the alpha/beta hydrolase family and that it c ontains a typical catalytic triad composed of Asp(211), His(241), and Ser(114). What is unusual is that the nucleophilic S-114 is not contai ned within the esterase consensus motif GXSXG although the stereochemi stry of the turn involving S-114 is almost identical to the nucleophil ic elbow found in alpha/beta hydrolases. In contrast to mammalian thio esterases, deacylation of LuxD was the rate-limiting step, with the le vel of acylated enzyme formed on reaction with myristoyl-CoA and the p re-steady-state burst of p-nitrophenol on cleavage of p-nitrophenyl my ristate 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 S-114 b y 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 re moved from the S114C mutant by neutral hydroxylamine, showing that a c ysteine residue had been acylated, Mutation of H-241 creating the doub le mutant, S114C . H241N, decreased acylation of the cysteine residue, These results provide direct kinetic and chemical evidence that S-114 is the site of acylation linked to H-241 in the charge relay system a nd have led to the recognition of a more general consensus motif flank ing the nucleophilic serine in thioesterases.