Functional role of a non-active site residue Trp23 on the enzyme activity of Escherichia coli thioesterase I/protease I/lysophospholipase L1

Escherichia coli possesses a versatile protein with the enzyme activities of thioesterase I, protease I, and lysophospholipase L1. 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 Ser10, Asp154, and His157 as a charge relay system, as well as Gly44 and Asn73 residues devoted to oxyanion hole stabilization. Geometrically, about 10 Å away from the enzyme catalytic cleft, Trp23 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 Trp23 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, Trp23 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.