Tryptophan Fluorescence of the lux-Specific Vibrio harveyi Acyl-ACP Thioesterase and Its Tryptophan Mutants: Structural Properties and Ligand-Induced Conformational Change

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• 作者：Jun Li ; Rose Szittner ; and Edward A. Meighen
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：November 17, 1998
• 年：1998
• 卷：37
• 期：46
• 页码：16130 - 16138
• 全文大小：126K
• 年卷期：v.37,no.46(November 17, 1998)
• ISSN：1520-4995

文摘

The lux-specific myristoyl-ACP thioesterase from Vibrio harveyi contains four tryptophanresidues, Trp23, Trp99, Trp186, and Trp213. Replacement of each of these residues with tyrosine bysite-directed mutagenesis coupled with fluorescence and quenching studies of the purified mutant andwild type thioesterases during catalysis has been used to probe ligand-induced conformational changes.Mutant W99Y retained high enzyme activity (80%) with W213Y and W23Y retaining intermediate activityand W186Y having the lowest activity (20%). The sum of the differential fluorescence spectra of theindividual tryptophans was identical to the fluorescence spectrum of the wild type thioesterase, showingthat mutation had not caused a major conformational change and energy transfer did not occur betweenthe tryptophans. Fluorescence emission maxima and quenching by acrylamide revealed that Trp213 andTrp23 are in a polar environment and/or exposed to solvent while Trp186 appeared to be buried insidethe molecule, consistent with the crystal structure of the thioesterase. The fluorescence intensities of thewild type, W23Y, W99Y, and W186Y thioesterases increased in direct correlation to their degree ofacylation with myristoyl-CoA, while the fluorescence of the acylated W213Y mutant remained constant,showing that the enhancement of fluorescence was entirely due to interaction of the acyl group withTrp213. Acrylamide quenching of the acylated mutants showed that the accessibility of the tryptophansto solvent was differentially altered and that the quenching of W23Y was enhanced in contrast to thequenching of the other mutants, supporting a ligand-induced conformational change during enzyme turnover.