Tryptophan fluorescence of the lux-specific Vibrio harveyi acyl-ACP thioesterase and its tryptophan mutants: structural properties and ligand-induced conformational change.

The lux-specific myristoyl-ACP thioesterase from Vibrio harveyi contains four tryptophan residues, Trp23, Trp99, Trp186, and Trp213. Replacement of each of these residues with tyrosine by site-directed mutagenesis coupled with fluorescence and quenching studies of the purified mutant and wild 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 activity and W186Y having the lowest activity (20%). The sum of the differential fluorescence spectra of the individual tryptophans was identical to the fluorescence spectrum of the wild type thioesterase, showing that mutation had not caused a major conformational change and energy transfer did not occur between the tryptophans. Fluorescence emission maxima and quenching by acrylamide revealed that Trp213 and Trp23 are in a polar environment and/or exposed to solvent while Trp186 appeared to be buried inside the molecule, consistent with the crystal structure of the thioesterase. The fluorescence intensities of the wild type, W23Y, W99Y, and W186Y thioesterases increased in direct correlation to their degree of acylation 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 with Trp213. Acrylamide quenching of the acylated mutants showed that the accessibility of the tryptophans to solvent was differentially altered and that the quenching of W23Y was enhanced in contrast to the quenching of the other mutants, supporting a ligand-induced conformational change during enzyme turnover.