Kim S, Narayana S V, Volanakis J E
Department of Medicine, University of Alabama at Birmingham 35294, USA.
J Immunol. 1995 Jun 1;154(11):6073-9.
We have investigated the structural determinants of the unique functional properties of complement factor D by constructing and testing a series of trypsin-like mutants of the enzyme. Mutational replacement of the primary substrate-binding pocket of factor D with that of trypsin resulted in a mutant (M1) with greatly reduced proteolytic activity and slightly reduced reactivity toward small thioester substrates. Combining the M1 mutations with substitution of Tyr for Ser94, previously shown to enhance substantially both the proteolytic and esterolytic activities of factor D, produced a mutant (M2) with reactivities similar to M1. Replacement of the surface loop formed by residues 184-188 of M1 and M2 with the corresponding loop of trypsin produced mutants exhibiting one and two orders of magnitude higher esterolytic activity, respectively, than native factor D. However, the proteolytic activity of both mutants was similar to that of M1 and M2. We conclude that loop184-188 is an important determinant of the geometry of the primary specificity pocket of factor D. The low proteolytic activity of these mutants supports the proposal that the proteolytically active conformation of factor D is induced by its natural substrate, C3bB.
我们通过构建和测试该酶的一系列类胰蛋白酶突变体，研究了补体因子D独特功能特性的结构决定因素。用胰蛋白酶的主要底物结合口袋对因子D进行突变替换，产生了一个突变体（M1），其蛋白水解活性大大降低，对小硫酯底物的反应性略有降低。将M1突变与用酪氨酸替代Ser94相结合（先前已证明这可大幅增强因子D的蛋白水解和酯水解活性），产生了一个反应性与M1相似的突变体（M2）。用胰蛋白酶的相应环替换M1和M2中由残基184 - 188形成的表面环，产生的突变体分别表现出比天然因子D高一个和两个数量级的酯水解活性。然而，这两个突变体的蛋白水解活性与M1和M2相似。我们得出结论，环184 - 188是因子D主要特异性口袋几何形状的重要决定因素。这些突变体的低蛋白水解活性支持了这样的提议，即因子D的蛋白水解活性构象是由其天然底物C3bB诱导产生的。
