Hirose Junzo, Kamigakiuchi Hiroshi, Iwamoto Hiroyuki, Fujii Hideaki, Nakai Masanori, Takenaka Mituru, Kataoka Rieko, Sugahara Makoto, Yamamoto Satoru, Fukasawa Kayoko M
Department of Applied Biological Science, Faculty of Life Science and Biotechnology, Fukuyama University, Gakuen-cho, Fukuyama 729-0292, Japan.
Arch Biochem Biophys. 2004 Nov 1;431(1):1-8. doi: 10.1016/j.abb.2004.07.033.
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.
二肽基肽酶III（DPP III）的锌结合基序（HELLGH）不同于金属肽酶常见的锌结合基序（HExxH）。为阐明DPP III的锌结合基序部分对酶活性的重要性，我们测定了在各种金属离子存在下，缺失亮氨酸（453）的脱辅基二肽基肽酶III（apo-Leu(453)-del-DPP III）的酶活性恢复情况，该酶具有与普通肽酶（HExxH）类似的基序（HELGH）。添加铜离子无法恢复缺失亮氨酸（453）的DPP III的酶活性，而添加铜离子可使脱辅基DPP III轻松重新激活。分离得到的Cu(II)-Leu(453)-del DPP III的可见光谱和电子顺磁共振光谱清楚地表明，Cu(II)-Leu(453)-del-DPP III的铜离子与基序部分（HELGH）结合，但未表现出任何酶活性。DPP III的基序部分直接影响DPP III铜衍生物中酶活性的表达。已确定一种完全不被DPP III消化的竞争性抑制剂，即组氨丙苯（His-Pro-Phe-His-Leu-d-Leu-Val-Tyr）。组氨丙苯对DPP III或Cu(II)-DPP III的抑制常数（K(i)）分别为4.1x10(-5)或3.8x10(-5)M。在竞争性肽抑制剂组氨丙苯存在下，Cu(II)-DPP III的电子顺磁共振光谱与Cu(II)-DPP III本身的光谱完全不同。这一结果清楚地表明，DPP III的金属离子位于活性位点并直接与底物相互作用。
