Kim S, Narayana S V, Volanakis J E
Department of Microbiology, University of Alabama at Birmingham 35294.
Biochemistry. 1994 Dec 6;33(48):14393-9. doi: 10.1021/bi00252a004.
Complement factor D is a serine protease with a single natural substrate, C3b-complexed factor B, and very low catalytic activity against synthetic esters. The recently solved X-ray crystal structure of factor D has demonstrated certain key differences from other serine protease in the conformation of residues of the catalytic triad and the substrate-binding regions. To investigate possible contributions of unique amino acid substitutions to these distinct structural and functional features of factor D, we constructed a series of mutants by substituting trypsin substrate-binding residues for the corresponding factor D residues. Wild-type and seven mutant factor D cDNAs were expressed stably in Chinese hamster ovary cells, and the recombinant proteins were purified from culture supernatants and assayed by hemolytic, proteolytic, and esterolytic assays. The combined results indicate that residues Thr-198, Ser-199, Arg-202, and perhaps also Val-203 provide determinants for substrate binding and catalysis. The data also provide additional support for the hypothesis that the proteolytically active conformation of the active center of factor D is induced by its substrate, C3bB.
补体因子D是一种丝氨酸蛋白酶，其唯一的天然底物是与C3b结合的因子B，对合成酯的催化活性非常低。最近解析的因子D的X射线晶体结构显示，在催化三联体残基和底物结合区域的构象方面，它与其他丝氨酸蛋白酶存在某些关键差异。为了研究独特的氨基酸取代对因子D这些独特的结构和功能特征可能产生的影响，我们构建了一系列突变体，用胰蛋白酶底物结合残基取代相应的因子D残基。野生型和七个突变型因子D cDNA在中国仓鼠卵巢细胞中稳定表达，重组蛋白从培养上清液中纯化出来，并通过溶血、蛋白水解和酯水解测定进行分析。综合结果表明，苏氨酸-198、丝氨酸-199、精氨酸-202以及可能还有缬氨酸-203的残基为底物结合和催化提供了决定因素。这些数据也为以下假设提供了额外支持：因子D活性中心的蛋白水解活性构象是由其底物C3bB诱导产生的。
