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 contai ns four tryptophan residues, Trp23, Trp99, Trp186, and Trp213. Replace ment of each of these residues with tyrosine by site-directed mutagene sis coupled with fluorescence and quenching studies of the purified mu tant and wild type thioesterases during catalysis has been used to pro be ligand-induced conformational changes. Mutant W99Y retained high en zyme activity (80%) with W213Y and W23Y retaining intermediate activit y and W186Y having the lowest activity (20%). The sum of the different ial fluorescence spectra of the individual tryptophans was identical t o the fluorescence spectrum of the wild type thioesterase, showing tha t mutation had not caused a major conformational change and energy tra nsfer did not occur between the tryptophans. Fluorescence emission max ima 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, W2 3Y, W99Y, and W186Y thioesterases increased in direct correlation to t heir degree of acylation with myristoyl-CoA, while the fluorescence of the acylated W213Y mutant remained constant, showing that the enhance ment 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 al tered and that the quenching of W23Y was enhanced in contrast to the q uenching of the other mutants, supporting a Ligand-induced conformatio nal change during enzyme turnover.