Conformational change analysis of thimet oligopeptidase

Thimet oligopeptidase (TOP, EC 3.4.24.15 ) is a metalloendopeptidase selective for peptides 5–17 amino acids in length. The tertiary structure is bilobal, with the active site located at the base of a deep substrate‐binding cleft that divides the enzyme. The enzyme is proposed to undergo a hinge‐bend structural change that is required for efficient substrate cleavage and could provide a mechanism for TOP's size restriction. Using a homology model of the substrate‐bound enzyme, we have identified charged residues lining opposite sides of the entrance to the active site cleft and designed several mutant forms of TOP to test the importance of these residues in stabilizing the closed form of the enzyme. Activity assays with quenched‐fluorescent substrates indicate a progressive decrease in activity as the charges of these residues are neutralized and then reversed. In addition, the susceptibility of enzyme activity and inhibitor binding to buffer ionic strength is altered significantly for the mutants when compared to WT TOP. Interestingly, a quenched‐fluorescent bradykinin substrate, which is cleaved irrespective of the conformational change, is also unaffected by the mutants. The data demonstrate that residues, near the surface of the enzyme, are crucial for enzyme activity and provide additional evidence for the substrate‐induced conformational changes in TOP. This study was made possible by the Saint Mary's College School of Science Summer Research Program with funding from the Robert W. and Beverly J. Summers Scholarships.