The metal-binding motif of dipeptidyl peptidase III directly influences the enzyme activity in the copper derivative of dipeptidyl peptidase III.
The zinc-binding motif (HELLGH) of dipeptidyl peptidase III (DPP III) is different from the common zinc-binding motif (HExxH) of metallopeptidases. To clarify the importance of the zinc-binding motif part of DPP III for enzymatic activity, we measured the recovery of the enzyme activity of apo-Leu(453)-deleted dipeptidyl peptidase III (apo-Leu(453)-del-DPP III), which has a motif (HELGH) like that of the common peptidase (HExxH), in the presence of various metal ions. The enzyme activity of apo-Leu(453)-deleted DPP III could not be recovered by the addition of cupric ions, while apo-DPP III could be easily reactivated by the addition of cupric ions. The visible and electron paramagnetic resonance spectra of the isolated Cu(II)-Leu(453)-del DPP III clearly show that the cupric ions of Cu(II)-Leu(453)-del-DPP III bound to the motif part (HELGH) but did not exhibit any enzyme activity. The motif part of DPP III directly influences the expression of the enzyme activity in the copper derivative of DPP III. The competitive inhibitor that is not at all digested by DPP III, Hisprophen (His-Pro-Phe-His-Leu-d-Leu-Val-Tyr), has been determined. The inhibition constant (K(i)) of Hisprophen for DPP III or Cu(II)-DPP III was 4.1x10(-5) or 3.8x10(-5)M, respectively. In the presence of the competitive peptide inhibitor, Hisprophen, the EPR spectra of Cu(II)-DPP III were completely different from that of Cu(II)-DPP III itself. This result clearly indicates that the metal ions of DPP III are located in the active site and directly interact with the substrate.
Department of Applied Biological Science, Faculty of Life Science and Biotechnology, Fukuyama University, Gakuen-cho, Fukuyama 729-0292, Japan. hirose@fubac.fukuyama-u.ac.jp <hirose@fubac.fukuyama-u.ac.jp>
Junzo
J
Hirose
Hiroshi
H
Kamigakiuchi
Hiroyuki
H
Iwamoto
Hideaki
H
Fujii
Masanori
M
Nakai
Mituru
M
Takenaka
Rieko
R
Kataoka
Makoto
M
Sugahara
Satoru
S
Yamamoto
Kayoko M
KM
Fukasawa
D020816:Amino Acid Motifs / Q000502:physiology; D020134:Catalytic Domain; D003300:Copper / Q000378:metabolism*; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D010446:Peptide Fragments; D003300:Copper; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C100152:dipeptidyl peptidase III; D015032:Zinc