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The Metal Binding Motif of Dipeptidyl Peptidase III Influences the Enzyme Act ivity in the Copper Derivative of Dipeptidyl Peptidase III.

二肽基肽酶 III 的金属结合基序影响二肽基肽酶 III 铜衍生物的酶活性。

基本信息

批准号：
14572042
负责人：
HIROSE Junzo
金额：
$ 1.86万
依托单位：
Fukuyama University, Department of Applied Biological Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

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 (HELLGH) of DPP III for the enzyme activity, we measured the recovery of the enzyme activity of apo-Leu^<453> 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> 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 EPR 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 didn't 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), have been found out. The inhibition constant (Ki) of Hisprophen for DPP III or Cu(II)-DPP III was 4. 1x10^<-5> 3.8xlO^<-5> M^<-1>, respectively. In the presence of the competitive peptide inhibitor, Hisprophen, the electron paramagnetic resonance (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 locate 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）不同于金属肽酶的常见锌结合基序（HExxH）。为了阐明DPP III的锌结合基序部分（HELLGH）对酶活性的重要性，我们测量了在各种金属离子存在下apo-Leu^<453>二肽基肽酶III（apo-Leu^<453>-del-DPP III）的酶活性恢复，该酶具有与普通肽酶（HExxH）相似的基序（HELGH）。apo-Leu~ DPP Ⅲ的酶活性<453>不能通过铜离子的加入而恢复，而apo-DPP Ⅲ则很容易通过铜离子的加入而被重新激活。分离的Cu（II）-Leu^ -del DPP III的可见光谱和EPR光谱<453>清楚地表明Cu（II）-Leu^ -del DPP III的二价铜离子<453>结合到基序部分（HELGH），但不显示任何酶活性。DPP III的基序部分直接影响DPP III的铜衍生物中酶活性的表达。已发现完全不被DPP III消化的竞争性抑制剂Hisprophen（His-Pro-Phe-His-Leu-d-Leu-Val-Tyr）。Hisprophen对DPP III或Cu（II）-DPP III的抑制常数（Ki）为4。1 × 10 - 4 ~ <-5>3.8 × 10 - 4 <-5>M-<-1>4。在竞争性肽抑制剂Hisprophen的存在下，Cu（II）-DPP III的电子顺磁共振（EPR）谱与Cu（II）-DPP III本身完全不同。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。