Gettins P, Sottrup-Jensen L
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
J Biol Chem. 1990 May 5;265(13):7268-72.
NMR and ESR spectroscopies have been used to examine the plasma protease inhibitor pregnancy zone protein (PZP) and its complex with chymotrypsin. The 1H NMR spectrum of PZP shows relatively few sharp resonances, which, by analogy with human alpha 2-macroglobulin, probably arise from the proteolytically sensitive bait region. Upon reaction with chymotrypsin to form a 1:1 protease.PZP tetramer complex, there is a large increase in the intensity of sharp resonances due to an increase in mobility of these residues. 35Cl NMR has been used to follow binding of zinc and manganese to apo-PZP. Zinc binding causes a linear broadening of the bulk Cl-, consistent with access of Cl- to PZP-bound zinc. Since zinc in the two highest affinity sites in human alpha 2-macroglobulin causes no broadening of Cl-, it is concluded that these zinc sites are absent from PZP. The mobility of chymotrypsin in the PZP.chymotrypsin complex was examined by covalently attaching a nitroxide spin label at the serine residue in the active site of the enzyme and examining the appearance of the ESR spectrum. The chymotrypsin is rigidly held by the PZP to which it is covalently bound. In an analogous experiment performed previously on alpha 2-macroglobulin, chymotrypsin, bound in the presence of methylamine and therefore largely noncovalently bound, was found to be free to rotate inside the cage formed by the protease inhibitor.
核磁共振（NMR）和电子自旋共振（ESR）光谱已被用于研究血浆蛋白酶抑制剂妊娠区蛋白（PZP）及其与胰凝乳蛋白酶的复合物。PZP的1H NMR光谱显示出相对较少的尖锐共振峰，通过与人类α2-巨球蛋白类比，这些共振峰可能源于对蛋白水解敏感的诱饵区域。与胰凝乳蛋白酶反应形成1:1蛋白酶 - PZP四聚体复合物后，由于这些残基的流动性增加，尖锐共振峰的强度大幅增加。35Cl NMR已被用于跟踪锌和锰与脱辅基PZP的结合。锌结合导致大量Cl-的线性展宽，这与Cl-接近与PZP结合的锌一致。由于人类α2-巨球蛋白中两个最高亲和力位点的锌不会导致Cl-展宽，因此得出结论，PZP中不存在这些锌位点。通过在酶活性位点的丝氨酸残基上共价连接一个氮氧化物自旋标记并检查ESR光谱的出现，研究了胰凝乳蛋白酶在PZP - 胰凝乳蛋白酶复合物中的流动性。胰凝乳蛋白酶被与其共价结合的PZP牢固地固定。在先前对α2-巨球蛋白进行的类似实验中，发现胰凝乳蛋白酶在甲胺存在下结合，因此主要是非共价结合，它可以在由蛋白酶抑制剂形成的笼内自由旋转。
