Nisimoto Y, Freeman J L, Motalebi S A, Hirshberg M, Lambeth J D
Department of Biochemistry, Aichi Medical University, Nagakute Aichi 480-11, Japan.
J Biol Chem. 1997 Jul 25;272(30):18834-41. doi: 10.1074/jbc.272.30.18834.
Activation of the respiratory burst oxidase involves the assembly of the membrane-associated flavocytochrome b558 with the cytosolic components p47(phox), p67(phox), and the small GTPase Rac. Herein, the interaction between Rac and p67(phox) is explored using functional and physical methods. Mutually facilitated binding (EC50) of Rac1 and p67(phox) within the NADPH oxidase complex was demonstrated using steady state kinetic methods measuring NADPH-dependent superoxide generation. Direct binding of Rac1 and Rac2 to p67(phox) was shown using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. An increase in the methylanthraniloyl fluorescence was seen with added p67(phox) but not p47(phox), and the emission maximum shifted from 445 to 440 nm. Rac1 and Rac2 bound to p67(phox) with a 1:1 stoichiometry and with Kd values of 120 and 60 nM, respectively. Mutational studies (Freeman, J., Kreck, M., Uhlinger, D. J., and Lambeth, J. D. (1994) Biochemistry 33, 13431-13435; Freeman, J. L., Abo, A., and Lambeth, J. D. (1996) J. Biol. Chem. 271, 19794-19801) previously identified two regions in Rac1 that are important for activity: the "effector region" (residues 26-45) and the "insert region" (residues 124-135). Proteins mutated in the effector region (Rac1(N26H), Rac1(I33N), and Rac1(D38N)) showed a marked increase in both the Kd and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding to p67(phox). Insert region mutations (Rac1(K132E) and L134R), while showing markedly elevated EC50 values, bound with normal affinity to p67(phox). The structure of Rac1 determined by x-ray crystallography reveals that the effector region and the insert region are located in defined sectors on the surface of Rac1. A model is discussed in which the Rac1 effector region binds to p67(phox), the C terminus binds to the membrane, and the insert region interacts with a different protein component, possibly cytochrome b558.
呼吸爆发氧化酶的激活涉及膜相关黄素细胞色素b558与胞质成分p47(phox)、p67(phox)以及小GTP酶Rac的组装。在此，使用功能和物理方法探索了Rac与p67(phox)之间的相互作用。利用测量NADPH依赖性超氧化物生成的稳态动力学方法，证明了NADPH氧化酶复合物中Rac1和p67(phox)的相互促进结合（EC50）。以结合到Rac上的GTP荧光类似物（甲基邻氨基苯甲酰基鸟苷-5'-[β,γ-亚氨基]三磷酸）作为报告基团，显示了Rac1和Rac2与p67(phox)的直接结合。加入p67(phox)时甲基邻氨基苯甲酰基荧光增强，而加入p47(phox)时则无增强，且发射最大值从445 nm移至440 nm。Rac1和Rac2与p67(phox)以1:1化学计量比结合，Kd值分别为120 nM和60 nM。突变研究（弗里曼，J.，克雷克，M.，乌林格，D. J.，和兰贝思，J. D.（1994年）《生物化学》33卷，13431 - 13435页；弗里曼，J. L.，阿博，A.，和兰贝思，J. D.（1996年）《生物化学杂志》271卷，19794 - 19801页）先前确定了Rac中对活性重要的两个区域：“效应器区域”（第26 - 45位氨基酸残基）和“插入区域”（第124 - 135位氨基酸残基）。效应器区域发生突变的蛋白质（Rac1(N26H)、Rac1(I33N)和Rac1(D38N)）的Kd和EC50均显著增加，表明该区域的突变通过抑制Rac与p67(phox)结合来影响活性。插入区域突变体（Rac1(K132E)和L134R）虽然EC50值显著升高，但与p67(phox)的结合亲和力正常。通过X射线晶体学确定的Rac1结构表明，效应器区域和插入区域位于Rac1表面的特定区域。文中讨论了一个模型，其中Rac1效应器区域与p67(phox)结合，C末端与膜结合，插入区域与不同的蛋白质成分相互作用，可能是细胞色素b558。
