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(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)。为了阐明DPP III的锌结合基序(HELLGH)对酶活性的重要性，我们测定了在不同金属离子存在下，apo-Leu^&lt；453&gt；二肽基肽酶III(apo-Leu^&lt；453&gt；-del-DPP III)的酶活性恢复率，该基序(HELGH)与普通多肽酶(HExxH)相似。加入铜离子不能恢复apo-Leu&lt；453&GT；DPP III的酶活力，而加入Cu2+容易使apo-DPP III重新激活。分离得到的Cu2+-Leu^&lt；453&gt；-del DPP III的可见光谱和EPR谱表明，Cu2+-Leu^&lt；453&gt；-del-DPP III与基序部分(HELGH)结合，但不显示任何酶活性。DPP III的基序部分直接影响酶活性在DPP III铜衍生物中的表达。目前已发现一种完全不被DPP III消化的竞争性抑制剂Hisproen(His-Pro-Phe-His-Leu-d-Leu-Val-Tyr)。Hisproen对DPP III和Cu(II)-DPP III的抑制常数分别为4.1×10~(-5)和3.8×10~(-5)×10~(-5)。在竞争性多肽抑制剂Hisproen存在下，Cu(II)-DPP III的电子顺磁共振(EPR)谱与其本身的电子顺磁共振(EPR)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中心，并直接与底物相互作用。

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.