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 (residues 450-455, 508) motif as the zinc binding site, is classified as a zinc metallopeptidase. The zinc dissociation constants of the wild type, Leu(453)-deleted, and E508D mutant of DPP III at pH 7.4 were 4.5 (+/-0.7) x 10(-13), 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 Co(2+), Ni(2+), and Cu(2+) ions completely recovered the enzyme activities as did Zn(2+). The dissociation constants of Co(2+), Ni(2+), and Cu(2+) 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 Co(2+)-DPP III was very similar to that of Co(2+)-carboxypeptidase A or Co(2+)-thermolysin, in which the Co(2+) is bound to two histidyl nitrogens, a water molecule, and a glutamate residue. The absorption spectrum of Cu(2+)-DPP III is also very similar to that of Cu(2+)-thermolysin. The EPR spectrum and the EPR parameters of Cu(2+)-DPP III were very similar to those of Cu(2+)-thermolysin but slightly different from those of Cu(2+)-carboxypeptidase A. The five lines of the superfine structure in the perpendicular region of the EPR spectrum in Cu(2+)-DPP III suggest that nitrogen atoms should coordinate to the cupric ion in Cu(2+)-DPP III. All of these data suggest that the donor set and the coordination geometry of the metal ions in DPP III, which has the HExxxH motif as the metal binding site, are very similar to those of the metal ions in thermolysin, which has the HExxH motif.
Hirose
J
J
Department of Applied Biological Science and Biotechnology, Faculty of Engineering, Fukuyama University, Gakuen-cho, Fukuyama 729-0292, Japan. hirose@fubac.fukuyama-u.ac.jp
Iwamoto
H
H
Nagao
I
I
Enmyo
K
K
Sugao
H
H
Kanemitu
N
N
Ikeda
K
K
Takeda
M
M
Inoue
M
M
Ikeda
T
T
Matsuura
F
F
Fukasawa
K M
KM
Fukasawa
K
K
D000818:Animals; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D011994:Recombinant Proteins; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III; D015032:Zinc