Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1

Escherichia coli TAP (thioesterase 1, EC 3.1.2.2) is a multifunctional enzyme with thioesterase, esterase, arylesterase, protease and lysophospholipase activities. Previous crystal structural analyses identified its essential amino acid residues as those that form a catalytic triad (Ser(10)-Asp(154)-His(157)) and those involved in forming an oxyanion hole (Ser(10)-Gly(44)-Asn(73)). To gain an insight into the biochemical roles of each residue, site-directed mutagenesis was employed to mutate these residues to alanine, and enzyme kinetic studies were conducted using esterase, thioesterase and amino- acid-derived substrates. Of the residues, His(157) is the most important, as it plays a vital role in the catalytic triad, and may also play a role in stabilizing oxyanion conformation. Ser(10) also plays a very important role, although the small residual activity of the S10A variant suggests that a water molecule may act as a poor substitute. The water molecule could poss-ibly be endowed with the nucleophilic-attacking character by His(157) hydrogen-bonding. Asp(154) is not as essential compared with the other two residues in the triad. It is close to the entrance of the substrate tunnel, therefore it predominantly affects substrate accessibility. Gly(44) plays a role in stabilizing the oxyanion intermediate and additionally in acyl-enzyme-intermediate transformation. N73A had the highest residual enzyme activity among all the mutants, which indicates that Asn(73) is not as essential as the other mutated residues. The role of Asn(73) is proposed to be involved in a loop(75-80) switch-move motion, which is essential for the accommodation of substrates with longer acyl-chain lengths.