Salivary amylase activity is dependent upon the conformation of a mobile loop

Amylases carry out the hydrolysis of (a)1,4-linked starch using a triad of catalytic residues Asp197, Glu233 and Asp300. Mutation of any of these residues abolishes the enzyme activity. Objectives: To engineer an inactive amylase without altering the catalytic triad. Methods: We examined the most flexible loop region in salivary amylase, residues 304-310, involved in substrate binding. Particularly, residues His305 and Gly306 reorient in the complex between amylase and acarbose to provide hydrogen bonding with bound glucose units. Residue Trp58 was selected as most appropriate to bring about a conformational change in the mobile loop since it is involved in a stacking interaction with residues His299 and His305, which are implicated in the enzyme mechanism. Using recombinant methods, we generated and determined the kinetic properties of the following mutants: Trp58Leu, D306 (lacking residues 306-310), Trp59Leu, His299Ala and His305Ala. Results: Kinetic analyses showed significant reduction (over 200-400 fold) in the specific activities for the mutants D306 and Trp58Leu while the other mutants showed only a 2-10 fold reduction. Comparison of the crystal structures of the mutants D306 and Trp58Leu with that of native amylase revealed that the His305 in both structures has moved significantly from the corresponding position in wild type structure. In addition, the mobile loop in the Trp58Leu mutant adopts an ordered structure. Conclusions: Starch or oligosaccharides may not be held in place in the active site during hydrolysis either due to the absence of the mobile loop in D306, or the adaptation of an ordered conformation in Trp58Leu. Interestingly, the catalytic triad in these mutants is intact and juxtaposed comparably with the native enzyme. These results strongly support that the segment 306-310 is critical in the binding and hydrolysis of starch substrates. This work was supported by USPHS Grant DE12585.