Interactions among residues CD3, E7, E10, and E11 in myoglobins: attempts to simulate the ligand-binding properties of Aplysia myoglobin.
Interactions among residues CD3, E7, E10, and E11 in myoglobins: attempts to simulate the ligand-binding properties of Aplysia myoglobin.
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肌红蛋白中残基 CD3、E7、E10 和 E11 之间的相互作用：尝试模拟海兔肌红蛋白的配体结合特性。
作者：
Smerdon,SJ;Krzywda,S;Brzozowski,AM;Davies,GJ;Wilkinson,AJ;Brancaccio,A;Cutruzzolá,F;Allocatelli,CT;Brunori,M;Li,T
修订稿于 1995 年 4 月 20 日收到®摘要：已在猪和抹香鲸肌红蛋白的赖氨酸/精氨酸 45 (CD3)、组氨酸 64 (E7)、苏氨酸 67 (E10) 和缬氨酸 68 (Ell) 残基上单独或组合引入定点突变。这些突变探究了这些关键远端口袋残基的作用，并代表了模拟海兔肌红蛋白血红素环境的尝试，该肌红蛋白在远端组氨酸不存在的情况下实现了中等高的 O2 亲和力。在软体动物肌红蛋白中，精氨酸-E10被认为摆动到血红素袋中并提供与结合的O 2 的氢键。 [0123] 氧和一氧化碳与H64V、T67A、T67V、T67E、T67R、V68I、V68T、H64V-T67R、H64V-V68T、H64V-V68I和H64V-T67R-V68I猪肌红蛋白突变体和T67R结合的缔合和解离速率常数，使用停流快速混合和闪光光解技术测量了抹香鲸肌红蛋白的 H64V-T67R 和 R45D-H64V-T67R 突变体。在猪或抹香鲸的肌红蛋白中用缬氨酸替代组氨酸-E7 会显着降低 O2 亲和力，同时增加 CO 亲和力。两个第二位点突变 T67R 和 V68T 增加了 H64V 突变体中的 O2 亲和力，即使单独引入时这些突变没有影响或分别降低了 Ko2。然而，H64V-T67R 突变体的氧亲和力比 A. limacina 肌红蛋白低 5-10 倍。猪H64VT67R双突变体的晶体结构揭示缬氨酸-E7侧链比软体动物蛋白中的更接近血红素平面～1A，这可能限制精氨酸-E10侧链进入血红素袋。 H64V-T67R 双突变体的 O2 亲和力不会因 R45D 替换而改变，但会因 V68I 突变而降低 10 倍。从 O2、CO 和 N3 结合以及 H64VT67R、H64V-V68I 和 H64V-V68T 双突变蛋白的晶体结构方面讨论了 T67R、V68I 和 V68T 突变与 H64V 取代的相互作用。在许多情况下，缬氨酸 64 背景中第二位点突变的影响与野生型背景中相应单突变所观察到的效果相反。这些结果可以通过配体缔合和解离的速率决定步骤的变化以及组氨酸64被缬氨酸取代后远端口袋水分子的损失来理解。
Revised Manuscript Received April 20, 1995® abstract: Site-directed mutations have been introduced singly and in combination at residues lysine/arginine45 (CD3), histidine64 (E7), threonine67 (E10), and valine68 (Ell) in pig and sperm whale myoglobins. The mutations probe the roles of these key distal pocket residuesand represent attempts to mimic the heme environment of Aplysia limacina myoglobin which achieves moderatelyhigh O2 affinity in the absence of a distal histidine. In the mollusc myoglobin, arginine-ElO is believed to swing into the heme pocket and provide a hydrogen bondto the bound O2. The association and dissociation rate constants for oxygen and carbon monoxide binding to H64V, T67A, T67V, T67E, T67R, V68I, V68T, H64V-T67R, H64V-V68T, H64V-V68I, and H64V-T67R-V68I pig myoglobin mutants and T67R, H64V-T67R, and R45D-H64V-T67R mutants of sperm whale myoglobin have been measured using stopped-flow rapid mixing and flashphotolysis techniques. Replacement of histidine-E7 with valine in either pig or sperm whale myoglobin drastically lowers O2 affinity while increasing CO affinity. Two second-site mutations, T67R and V68T, increase O2 affinity in the H64V mutant, even though when introduced singly these mutations have no effect or lower Ko2, respectively. However, the oxygen affinities of the H64V-T67R mutants are 5-10-fold lower than that of A. limacina myoglobin. The crystal structure of the pig H64VT67R doublemutant reveals that the valine-E7 side chain is~ 1A closer to the heme plane than in the mollusc protein which may restrict access of the arginine-ElO side chaininto the heme pocket. The O2 affinity of the H64V-T67R double mutant is notaltered by the R45D replacement but is reduced 10-fold by the V68I mutation. The interactive effects of the T67R, V68I, and V68T mutations with the H64V substitution are discussed in terms of O2, CO, and N3-binding and the crystal structures of the H64VT67R, H64V-V68I, and H64V-V68T double-mutant proteins. In many instances, the effects of secondsite mutationsin the valine64 background are the opposite of those observed for the corresponding single mutations in the wild type background. These results can be understood in terms of the changes in the rate-determining steps for ligand association and dissociation and the loss of distal pocket water molecules which follow replacement of histidine64 by valine.