Characterization of the metal-substituted dipeptidyl peptidase III (rat liver)

Dipeptidyl peptidase III (DPP III) (EC 3.4.14.4), which has a HELLGH-E (res idues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissociation constants of the wild type, Leu(45 3)-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (+/-0.7) x 10(-1 3), 5.8 (+/-0.7) x 10(-12), and 3.2 (+/-0.9) x 10(-10) M, respectively. The recoveries of the enzyme activities by the addition of various metal ions to apo-DPP III were also measured, and Co2+, Ni2+, and Cu2+ ions completely recovered the enzyme activities as did Zn2+. The dissociation constants of Co2+, Ni2+, and Cu2+ ions for apo-DPP III at pH 7.4 were 8.2 (+/-0.9) x 10 (-13), 2.7 (+/-0.3) x 10(-12), and 1.1 (+/-0.1) x 10(-14) M, respectively. The shape of the absorption spectrum of CO2+-DPP III was very similar to th at of CO2+-carboxypeptidase A or CO2+-thermolysin, in which the CO2+ is bou nd to two histidyl nitrogens, a water molecule, and a glutamate residue. Th e absorption spectrum of Cu2+-DPP III is also very similar to that of Cu2+- thermolysin. The EPR spectrum and the EPR parameters of Cu2+-DPP III were v ery similar to those of Cu2+-thermolysin but slightly different from those of Cu2+-carboxypeptidase A. The five lines of the superfine structure in th e perpendicular region of the EPR spectrum in Cu2+-DPP III suggest that nit rogen atoms should coordinate to the cupric ion in Cu2+-DPP III. All of the se data suggest that the donor set and the coordination geometry of the met al ions in DPP III, which has the HExxxH motif as the metal binding site, a re very similar to those of the metal ions in thermolysin, which has the HE xxH motif.