Functional significance of the conformational dynamics of the N-terminal segment of secreted phospholipase A2 at the interface.
Functional significance of the conformational dynamics of the N-terminal segment of secreted phospholipase A2 at the interface.
复制标题
分泌型磷脂酶 A2 N 端片段在界面处的构象动力学的功能意义。
DOI：
10.1021/bi00181a010
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Jain,MK
中科院分区：
文献类型：
--
作者：
Maliwal,BP;Yu,BZ;Szmacinski,H;Squier,T;vanBinsbergen,J;Slotboom,AJ;Jain,MK
修订稿于 1993 年 12 月 10 日收到®摘要：对几种猪胰磷脂酶 A2 (PLA2) 的动力学和荧光特性进行了表征，这些猪胰磷脂酶 A2 (PLA2) 在 N 末端区域以及酪氨酸 52 和 73 发生了取代和缺失。取代 Ala-1D-Ala 或-Gly、Trp-3-Phe、Gln-4-Nle、Arg-6-Glu、Tyr-52-Phe 和 Tyr-73-Phe 对它们以高亲和力结合的阴离子界面上的界面催化活性至多仅具有适度的影响。观察到，N-末端区域缺乏一个或多个连续残基的缺失突变体在快速模式下的水解率降低了50-95%。对缺乏 Ala-1 (des-l-AMPA) 的缺失突变体的详细动力学分析表明，速率降低 50% 是由于界面 Michaelis-Menten 参数 Km* 增加了 5 倍，而 kM 没有显着变化。这些结果和直接测量表明，Ala-1 缺失的主要作用是降低对活性位点定向配体的亲和力。尽管这些突变体对阴离子界面的亲和力与野生型相同，但对两性离子中性稀释剂的亲和力却相当低。这些酶的荧光量子产率的显着差异和频域荧光强度衰减的异质性表明，在溶液中和界面处，N端区域是构象的集合而不是离散状态。其他结果表明，des-l-AMPA 中 Trp-3 的界面微环境极性更强，Trp-3 更容易被丙烯酰胺猝灭。这表明在 des-l-AMPA 的情况下微界面的去溶剂化不完全。 Trp-3 的频域各向异性衰减表明游离 PLA2 的 N 端区域有些刚性，并且随着 Ala-1 和 Leu-2 的删除而变得灵活。另一方面，在 DTPM 囊泡上，PLA2 和 des-l-AMPA 中的 N 末端区域基本上是刚性的，并且在结合的 des-1, 2-AMPA 中仅具有适度的柔性。这些观察结果证明了PLA2的E*I形式的N端区域的配体诱导的刚性化，并且令人惊讶的是在不存在Ala-1的情况下观察到这种刚性化。总而言之，这些结果表明，Ala-1 和相关的氢键网络通过其在界面去溶剂化中的作用影响活性位点定向配体的结合，但对催化周转循环的化学步骤没有显着影响。
Revised Manuscript Received December 10, 1993® abstract: The kinetic and fluorescence properties of several pig pancreatic phospholipase A2 (PLA2) with substitutions and deletions in the N-terminal region and of tyrosines 52 and 73 are characterized. The substitutions Ala-lD-Ala or-Gly, Trp-3-Phe, Gln-4-Nle, Arg-6-Glu, Tyr-52-Phe, and Tyr-73-Phe had at the most only a modest effect on the interfacial catalytic activity on the anionic interface to which they bind with high affinity. The observed rate of hydrolysis in the scooting mode by deletion mutants lacking one or more successive residues from the N-terminal region was lower by 50-95%. Detailed kinetic analysis of the deletion mutant lacking Ala-1 (des-l-AMPA) showed that the 50% decrease in the rate is dueto a 5-fold increase in the interfacial Michaelis-Menten parameter, Km*, without a significant change in kM. These results and direct measurements show that the primary effect of Ala-1 deletion is to lower the affinity for the active site directed ligands. Although the affinity of these mutants for anionic interface remains the same as for the wild type, the affinity for zwitterionic neutral diluents is considerably lower. Significant differences in the fluorescence quantum yields and the heterogeneity in the frequency-domain fluorescence intensity decays of these enzymes suggest that both in solution and at the interface the N-terminal region is an ensemble of conformations rather than a discrete state. Additional results suggest that the interfacial microenvironment of Trp-3in des-l-AMPA is more polar and Trp-3 is more accessible to quenching by acrylamide. This indicates incomplete desolvation of the microinterface in case of des-l-AMPA. The frequency-domain anisotropy decays of Trp-3 indicate that the N-terminal region of free PLA2 is somewhat rigid and becomes flexible as Ala-1 and Leu-2 are deleted. On the other hand, on DTPM vesicles, the N-terminal region is essentially rigid in PLA2 and des-l-AMPA, and it is only modestly flexible in bound des-1, 2-AMPA. These observations demonstrate a ligand-induced rigidification of the N-terminal region of the E* I form of PLA2, and it is surprising that such a rigidification is observed in the absence of Ala-1. Taken together these results show that Ala-1 and the associated hydrogen-bond network affect the binding of an active site directed ligand via its role in desolvation of the interface butwithout a significant effect on the chemical step of the catalytic turnover cycle.