Adachi K, Konitzer P, Paulraj C G, Surrey S
Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine 19104.
J Biol Chem. 1994 Jul 1;269(26):17477-80.
X-ray crystallographic studies indicate that the hydrophobic acceptor pocket made by E and F helices involving Leu-beta 88 and Phe-beta 85 is critical for the formation of stable hydrophobic interactions with Val-beta 6 on an adjacent deoxy-hemoglobin (Hb) S tetramer. Ala and Phe substitutions at the beta 88 position in Hb S were made using a yeast expression system in an effort to clarify the role of Leu-beta 88 in creating a suitable acceptor site for Val-beta 6 during polymerization of Hb S. Both Ala- and Phe-beta 88 substitutions in Hb S inhibited polymerization compared with Hb S. Critical concentrations for polymerization of alpha 2 beta 2 Val-6,Ala-88 and alpha 2 beta 2Val-6,Phe-88 were 6- and 10-fold higher, respectively, than that of Hb S (alpha 2 beta 2Val-6,Leu-88). Deoxy-Hb S containing Phe-beta 88 polymerized without a delay time like Trp-beta 6- and Phe-beta 6-substituted hemoglobins (Adachi, K., Konitzer, P., Kim, J., Welch, N., and Surrey, S. (1993) J. Biol. Chem. 268, 21650-21656). In contrast, oversaturated deoxy-Hb S containing Ala-beta 88 also polymerized without a delay time; however, with decreasing hemoglobin concentrations, the kinetics of polymerization were biphasic. At lower hemoglobin concentrations, closer to the critical concentration for polymerization, deoxy-Hb S containing Ala-beta 88 polymerized after a distinct delay time. These results suggest that bulky beta 88 hydrophobic replacements like Phe may sterically inhibit insertion of Val-beta 6 into the acceptor pocket. In contrast, smaller sized, less hydrophobic amino acids like Ala compared with Leu-beta 88 may allow insertion of Val-beta 6 into the acceptor pocket but may not promote stable protein-protein interactions with an adjacent Hb molecule. Stereospecificity and hydrophobicity of the Val-beta 6 hydrophobic acceptor pocket as well as the beta 6 amino acid are, therefore, critical for polymerization of deoxy-Hb S.
X射线晶体学研究表明，由E螺旋和F螺旋形成的、涉及亮氨酸-β88和苯丙氨酸-β85的疏水受体口袋，对于与相邻的脱氧血红蛋白（Hb）S四聚体上的缬氨酸-β6形成稳定的疏水相互作用至关重要。利用酵母表达系统对Hb S中β88位点进行丙氨酸和苯丙氨酸取代，以阐明亮氨酸-β88在Hb S聚合过程中为缬氨酸-β6创建合适受体位点的作用。与Hb S相比，Hb S中的丙氨酸-β88和苯丙氨酸-β88取代均抑制了聚合反应。α2β2缬氨酸-6、丙氨酸-88和α2β2缬氨酸-6、苯丙氨酸-88聚合的临界浓度分别比Hb S（α2β2缬氨酸-6、亮氨酸-88）高6倍和10倍。含有苯丙氨酸-β88的脱氧Hb S像色氨酸-β6和苯丙氨酸-β6取代的血红蛋白一样无延迟时间地聚合（安达千春、科尼策、金、韦尔奇和萨里，《生物化学杂志》，第268卷，第21650 - 21656页，1993年）。相比之下，含有丙氨酸-β88的过饱和脱氧Hb S也无延迟时间地聚合；然而，随着血红蛋白浓度降低，聚合动力学呈双相性。在较低血红蛋白浓度下，更接近聚合临界浓度时，含有丙氨酸-β88的脱氧Hb S在明显延迟时间后聚合。这些结果表明，像苯丙氨酸这样的大体积β88疏水取代可能在空间上抑制缬氨酸-β6插入受体口袋。相比之下，与亮氨酸-β88相比，像丙氨酸这样体积较小、疏水性较低的氨基酸可能允许缬氨酸-β6插入受体口袋，但可能无法促进与相邻Hb分子的稳定蛋白质-蛋白质相互作用。因此，缬氨酸-β6疏水受体口袋以及β6氨基酸的立体特异性和疏水性对于脱氧Hb S的聚合至关重要。
