Horowitz P M, Bowman S
University of Texas Health Science Center at San Antonio, Department of Biochemistry 78284.
J Biol Chem. 1987 Oct 25;262(30):14544-8.
For the first time, the enzyme rhodanese has been proteolytically cleaved to give species that most likely correspond to individual domains. This indicates cleavage can occur in the interdomain tether. Further, the conditions for cleavage show that availability of the susceptible bond(s) depends on conformational changes triggered by oxidative inactivation. Rhodanese, without persulfide sulfur (E), was oxidized consequent to incubation with phenylglyoxal, NADH, or hydrogen peroxide. The oxidized enzyme (Eox) was probed using the proteolytic enzymes endoproteinase glutamate C (V8), trypsin, chymotrypsin, or subtilisin. The proteolytic susceptibility of Eox, formed using hydrogen peroxide, was compared with that of E and the form of the enzyme containing transferred sulfur, ES. ES was totally refractory to proteolysis, while E was only clipped to a small extent by trypsin or V8 and not at all by chymotrypsin or subtilisin. Eox was susceptible to proteolysis by all the proteases used, and, although there were some differences among the proteolytic patterns, there was always a band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to Mr = 16,500. This was the only band observed in addition to the parent species (Mr = 33,000) when Eox was digested with chymotrypsin, and conservation of total protein was observed after digestion up to 90 min. No additional species were observable on silver staining, although there was some indication that the band at 16,500 might be a doublet. The results are consistent with the occurrence of a conformational change after oxidation that results in increased exposure and/or flexibility of the interdomain tether which contains residues that meet the specificity requirements of the proteases used.
首次实现了对硫氰酸酶进行蛋白水解切割，得到了很可能对应于各个结构域的片段。这表明切割可发生在结构域间的连接区。此外，切割条件表明，敏感键的可用性取决于氧化失活引发的构象变化。不含过硫化物硫的硫氰酸酶（E）在与苯乙二醛、NADH或过氧化氢孵育后被氧化。使用蛋白水解酶谷氨酸内切蛋白酶C（V8）、胰蛋白酶、胰凝乳蛋白酶或枯草杆菌蛋白酶对氧化后的酶（Eox）进行检测。将用过氧化氢形成的Eox的蛋白水解敏感性与E以及含有转移硫的酶形式ES进行比较。ES对蛋白水解完全耐受，而E仅被胰蛋白酶或V8轻微切割，未被胰凝乳蛋白酶或枯草杆菌蛋白酶切割。Eox对所有使用的蛋白酶都敏感，尽管蛋白水解模式存在一些差异，但在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上总有一条对应于Mr = 16,500的条带。当用胰凝乳蛋白酶消化Eox时，这是除亲本物种（Mr = 33,000）外观察到的唯一一条带，并且在消化长达90分钟后观察到总蛋白守恒。银染未观察到其他条带，尽管有迹象表明16,500处的条带可能是双重条带。结果与氧化后发生构象变化一致，该变化导致结构域间连接区的暴露增加和/或灵活性增加，该连接区含有符合所用蛋白酶特异性要求的残基。
