Identification of an Additional Metal-Binding Site in Human Dipeptidyl Peptidase III

Dipeptidyl peptidase III (DPP III, EC 3.4.14.4) is a monozinc metalloexopeptidase that hydrolyzes dipeptides from the N-terminus of peptides consisting of three or more amino acids. Recently, DPP III has attracted great interest from scientists, and numerous studies have been conducted showing that it is involved in the regulation of various physiological processes. Since it is the only metalloenzyme among the dipeptidyl peptidases, we considered it important to study the process of binding and exchange of physiologically relevant metal dications in DPP III. Using fluorimetry, we measured the K(d) values for the binding of Zn(2+), Cu(2+), and Co(2+) to the catalytic site, and using isothermal titration calorimetry (ITC), we measured the K(d) values for the binding of these metals to an additional binding site. The structure of the catalytic metal’s binding site is known from previous studies, and in this work, the affinities for this site were calculated for Zn(2+), Cu(2+), Co(2+), and Mn(2+) using the QM approach. The structures of the additional binding sites for the Zn(2+) and Cu(2+) were also identified, and MD simulations showed that two Cu(2+) ions bound to the catalytic and inhibitory sites exchanged less frequently than the Zn(2+) ions bound to these sites.