Perlman R K, Gehm B D, Kuo W L, Rosner M R
Ben May Institute, University of Chicago, Illinois 60637.
J Biol Chem. 1993 Oct 15;268(29):21538-44.
Insulin-degrading enzyme (IDE), a nonlysosomal metalloprotease involved in metabolizing internalized insulin, has catalytic properties that have been strongly conserved through evolution. Two major properties distinguish IDE from the prototypic metalloprotease thermolysin. 1) It is inhibited by cysteine protease inhibitors as well as metalloprotease inhibitors; 2) it contains an inversion of the HEXXH active site motif of thermolysin, where the histidines coordinate zinc and the glutamate participates in catalysis. Furthermore, cysteine is adjacent to the glutamate residue (HXCEH) in human, rat, and Drosophila IDE, although it is not conserved in their close homologue, Escherichia coli protease III. This cysteine has been postulated to mediate the differential sensitivity of IDE and protease III to cysteine protease inhibitors and chelators. The role of the cysteine in IDE catalysis and inhibitor sensitivity was examined by mutating Cys110 to glycine or serine. To determine whether glutamate in this unusual motif participates in catalysis, we mutated Glu111 to aspartate, valine, or glutamine. Vectors containing wild type or mutant enzymes were transfected into COS cells, and expression was confirmed by Western blotting. Although the glutamate mutants were devoid of insulin degrading activity, the cysteine mutants were indistinguishable from wild type enzyme in both catalytic activity and sensitivity to inhibitors. The loss of activity in the glutamate mutants was not due to gross alterations in tertiary structure, as shown by retention of the ability to bind substrate and by conservative and nonconservative mutation of a neighboring residue with no apparent effect on catalysis. These results demonstrate that the conserved glutamate in the zinc-binding site of human insulin-degrading enzyme is a major catalytic residue, while a conserved cysteine in this region is not essential for catalysis or inhibitor sensitivity.
胰岛素降解酶（IDE）是一种参与内化胰岛素代谢的非溶酶体金属蛋白酶，其催化特性在进化过程中得到了强烈的保留。有两个主要特性将IDE与原型金属蛋白酶嗜热菌蛋白酶区分开来。1）它受到半胱氨酸蛋白酶抑制剂以及金属蛋白酶抑制剂的抑制；2）它包含嗜热菌蛋白酶HEXXH活性位点基序的倒置，其中组氨酸与锌配位，谷氨酸参与催化。此外，在人、大鼠和果蝇的IDE中，半胱氨酸与谷氨酸残基相邻（HXCEH），尽管在它们的近亲同源物大肠杆菌蛋白酶III中并不保守。据推测，这个半胱氨酸介导了IDE和蛋白酶III对半胱氨酸蛋白酶抑制剂和螯合剂的不同敏感性。通过将Cys110突变为甘氨酸或丝氨酸，研究了半胱氨酸在IDE催化和抑制剂敏感性中的作用。为了确定这个不寻常基序中的谷氨酸是否参与催化，我们将Glu111突变为天冬氨酸、缬氨酸或谷氨酰胺。将含有野生型或突变型酶的载体转染到COS细胞中，并通过蛋白质印迹法确认表达。尽管谷氨酸突变体没有胰岛素降解活性，但半胱氨酸突变体在催化活性和对抑制剂的敏感性方面与野生型酶没有区别。谷氨酸突变体活性的丧失不是由于三级结构的总体改变，这表现为保留了结合底物的能力以及相邻残基的保守和非保守突变对催化没有明显影响。这些结果表明，人胰岛素降解酶锌结合位点中保守的谷氨酸是主要的催化残基，而该区域中保守的半胱氨酸对催化或抑制剂敏感性不是必需的。
