Lai H H, Li T, Lyons D S, Phillips G N, Olson J S, Gibson Q H
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892, USA.
Proteins. 1995 Aug;22(4):322-39. doi: 10.1002/prot.340220404.
The role of Phe-46(CD4) in modulating the functional properties of sperm whale myoglobin was investigated by replacing this residue with Leu, Ile, Val, Ala, Trp, Tyr, and Glu. This highly conserved amino acid almost makes direct contact with the distal histidine and has been postulated to affect ligand binding. The overall association rate constants for CO, O2, and NO binding were little affected by decreasing the size of residue 46 step-wise from Phe to Leu to Val to Ala. In contrast, the rates of CO, O2, and NO dissociation increased 4-, 10-, and 25-fold, respectively, for the same series of mutants, causing large decreases in the affinity of myoglobin for all three diatomic gases. The rates of autooxidation at 37 degrees C, pH 7.0 increased dramatically from approximately 0.1-0.3 h-1 for wild-type, Tyr-46, and Trp-46 myoglobins to 1.5, 5.2, 4.9, and 5.0 h-1 for the Leu-46, Ile-46, Val-46 and Ala-46 mutants, respectively. Rates of NO and O2 geminate recombination were measured using 35 ps and 9 ns laser excitation pulses. Decreasing the size of residue 46 causes significant decreases in the extent of both picosecond and nanosecond rebinding processes. High resolution structures of Leu-46 and Val-46 metmyoglobins, Val-46 CO-myoglobin, and Val-46 deoxymyoglobin were determined by X-ray crystallography. When Phe-46 is replaced by Val, the loss of internal packing volume is compensated by (1) contraction of the CD corner toward the core of the protein, (2) movement of the E-helix toward the mutation site, (3) greater exposure of the distal pocket to intruding solvent molecules, and (4) large disorder in the position of the side chain of the distal histidine (His-64). In wild-type myoglobin, the van der Waals contact between C zeta of Phe-46 and C beta of His-64 appears to restrict rotation of the imidazole side chain. Insertion of Val at position 46 relieves this steric restriction, allowing the imidazole side chain to rotate about the C alpha - C beta bond toward the surface of the globin and about the C beta - C gamma bond toward the space previously occupied by the native Phe-46 side chain. This movement disrupts hydrogen bonding with bound ligands, causing significant decreases in affinity, and opens the distal pocket to solvent water molecules, causing marked increases in the rate of autooxidation.(ABSTRACT TRUNCATED AT 400 WORDS)
通过用亮氨酸、异亮氨酸、缬氨酸、丙氨酸、色氨酸、酪氨酸和谷氨酸取代第46位苯丙氨酸（CD4），研究了其在调节抹香鲸肌红蛋白功能特性中的作用。这个高度保守的氨基酸几乎与远端组氨酸直接接触，据推测会影响配体结合。从苯丙氨酸到亮氨酸再到缬氨酸最后到丙氨酸逐步减小第46位残基的大小，对CO、O2和NO结合的总体缔合速率常数影响不大。相反，对于同一系列的突变体，CO、O2和NO的解离速率分别增加了4倍、10倍和25倍，导致肌红蛋白对所有三种双原子气体的亲和力大幅下降。在37摄氏度、pH 7.0条件下，野生型、第46位酪氨酸和第46位色氨酸的肌红蛋白的自氧化速率约为0.1 - 0.3 h-1，而第46位亮氨酸、第46位异亮氨酸、第46位缬氨酸和第46位丙氨酸的突变体的自氧化速率分别增加到1.5、5.2、4.9和5.0 h-1。使用35皮秒和9纳秒的激光激发脉冲测量了NO和O2的双分子复合速率。减小第46位残基的大小会导致皮秒和纳秒重结合过程的程度显著降低。通过X射线晶体学确定了第46位亮氨酸和第46位缬氨酸高铁肌红蛋白、第46位缬氨酸CO - 肌红蛋白和第46位缬氨酸脱氧肌红蛋白的高分辨率结构。当第46位苯丙氨酸被缬氨酸取代时，内部堆积体积的损失通过以下方式得到补偿：（1）CD角向蛋白质核心收缩；（2）E螺旋向突变位点移动；（3）远端口袋更易暴露于侵入的溶剂分子；（4）远端组氨酸（His - 64）侧链位置的大量无序。在野生型肌红蛋白中，第46位苯丙氨酸的Cζ与His - 64的Cβ之间的范德华接触似乎限制了咪唑侧链的旋转。在第46位插入缬氨酸可缓解这种空间限制，使咪唑侧链能够围绕Cα - Cβ键向球蛋白表面旋转，并围绕Cβ - Cγ键向先前被天然苯丙氨酸 - 46侧链占据的空间旋转。这种移动破坏了与结合配体的氢键，导致亲和力显著下降，并使远端口袋向溶剂水分子开放，导致自氧化速率显著增加。（摘要截取自400字）
