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

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• 作者：Jun Li ; Rose Szittner ; Zygmunt S. Derewenda ; and Edward A. Meighen
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：August 6, 1996
• 年：1996
• 卷：35
• 期：31
• 页码：9967 - 9973
• 全文大小：420K
• 年卷期：v.35,no.31(August 6, 1996)
• ISSN：1520-4995

文摘

The lux-specific myristoyl-ACP thioesterase (LuxD) is responsiblefor diverting myristic acidinto the luminescent system and can function as an esterase andtransferase as well as cleave myristoyl-CoA and other thioesters. The recently elucidated crystal structureof the enzyme shows that it belongsto the / hydrolase family and that it contains a typicalcatalytic triad composed of Asp²¹¹, His²⁴¹,andSer¹¹⁴. What is unusual is that the nucleophilicS¹¹⁴ is not contained within the esterase consensusmotifGXSXG although the stereochemistry of the turn involvingS¹¹⁴ is almost identical to the nucleophilicelbow found in / hydrolases. In contrast to mammalianthioesterases, deacylation of LuxD was therate-limiting step, with the level of acylated enzyme formed onreaction with myristoyl-CoA and thepre-steady-state burst of p-nitrophenol on cleavage ofp-nitrophenyl myristate both being 0.7 mol/mol.Cold chase experiments showed that the deacylation rate of LuxDcorresponded closely to the turnoverrate of the enzyme with ester or thioester substrates. Replacementof S¹¹⁴ by a cysteine residue generateda mutant (S114C) that was acylated with the same pH dependence as LuxDbut had greatly diminishedcapacity to transfer acyl groups to water or glycerol. The acylgroup could be removed from the S114Cmutant by neutral hydroxylamine, showing that a cysteine residue hadbeen acylated. Mutation of H²⁴¹creating the double mutant, S114C·H241N, decreased acylation ofthe cysteine residue. These resultsprovide direct kinetic and chemical evidence that S¹¹⁴ isthe site of acylation linked to H²⁴¹ in thechargerelay system and have led to the recognition of a more generalconsensus motif flanking the nucleophilicserine in thioesterases.