Escherichia coli possesses a versatile protein with the enzyme activities of thioesterase I, protease I, and lysophospholipase L(1). The protein is dubbed as TAP according to the chronological order of gene discovery (TesA/ApeA/PldC). Our previous studies showed that TAP comprises the catalytic triad Ser(10), Asp(154), and His(157) as a charge relay system, as well as Gly(44) and Asn(73) residues devoted to oxyanion hole stabilization. Geometrically, about 10 A away from the enzyme catalytic cleft, Trp(23) showed a stronger resonance shift than the backbone amide resonance observed in the nuclear magnetic resonance (NMR) analyses. In the present work, we conducted site-directed mutagenesis to change Trp into alanine (Ala), phenylalanine (Phe), or tyrosine (Tyr) to unveil the role of the Trp(23) indole ring. Biochemical analyses of the mutant enzymes in combination with TAP's three-dimensional structures suggest that by interlinking the residues participating in this catalytic machinery, Trp(23) could effectively influence substrate binding and the following turnover number. Moreover, it may serve as a contributor to both H-bond and aromatic-aromatic interaction in maintaining the cross-link within the interweaving framework of protein.