Selective protein cleavage reagents carrying metal chelates moiety : Synthesis and application to the structural analysis of trypsin.

在我们之前的工作中，我们发现苯并脒衍生物对胰蛋白酶和胰蛋白酶样酶具有很强的结合亲和力。作为工作的延伸，我们设计了含有金属螯合基团和氨基基团的化合物，以期获得胰蛋白酶和胰蛋白酶样酶的选择性修饰试剂。这些试剂被期望在金属在酶的结合位点得到紧密接触的区域介导酶主链的裂解。酶试剂复合物的三维结构。胰蛋白酶肽键裂解试剂。1997年，我们合成了38种螯合物作为胰蛋白酶特异性裂解试剂的候选物，并测定了它们对胰蛋白酶和胰蛋白酶样酶的结合亲和力。经分析，它们对这些酶具有很强的结合亲和力，Ki值范围为10^5 - 10^ 6 Mi。在1998财政年度期间，我们研究了这些螯合物介导的介导反应。当酶与试剂在过氧化氢和抗坏血酸存在下孵育时，观察到裂解。用SDS- PAGE分析了由于键断裂引起的肽片段。结果表明，裂解发生在Gly195 -A1a2O4内的区域。结果表明，该区域在三维结构上接近酶的活性位点。

In our previous work, it was found that benzamidine derivatives behave as strong binding affinity towards trypsin and trypsin-like enzymes. As an extension of the wurk, we designed compounds which contained metal chelate moiety as well as the amidine group with a view to obtaining selective modification reagents for trypsin and trypsin-like enzymes. The reagents were expected to mediate cleavage of the enzyme backbone at the region where the metal attained close contact when they were bound in the binding site of the enzyme. the three-dimensional structure of the enzyme-reagent complex. eaction peptide bond cleavage reagents for trypsin.In 1997 we synthesized 38 chelates as candidate for trypsin-specific cleavage reagent, and their binding affinity toward trypsin and trypsin-like enzymes was determinecL They were analyzed to exhibit strong binding affinity toNard these enzymes with Ki values range of 10^5 - 1O^6 Mi. During the period of the fiscal year of 1998 the medification reaction mediated by these chelates were investigated. Cleavage was observed when the enzyme was incubated with the reagent in the presence of hydrogenperoxide and ascorbate. A peptide fragment due to bond cleavage was analyzed by SDS- PAGE.It was suggested that the cleavage occurred at the region within Gly195 -A1a2O4. It was concluded that the region is close to the active site of the enzyme in its three-dimensional structure.