Thewes T, Constantine K, Byeon I J, Llinás M
Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890.
J Biol Chem. 1990 Mar 5;265(7):3906-15.
The binding of small molecules to the kringle 5 domain fragment of human plasminogen has been investigated by 1H NMR spectroscopy at 300 MHz. The compounds tested as potential ligands include L-arginine, L-lysine, and a number of aliphatic and aromatic analogs of similar size but different ionic charge configurations. Ligand/kringle 5 association constant (Ka) values were obtained from ligand titration experiments at 22 degrees C, pH 7.2. Neither L-arginine nor N alpha-acetyl-L-arginine and N alpha-acetyl-L-arginine methyl ester bind measurably to kringle 5 (Ka approximately less than 0.05 mM-1). In contrast, binding of hexylamine or epsilon-aminocaproic acid (epsilon ACA) is favored (Ka approximately 2.9 and 10.5 mM-1, respectively). Benzamidine and p-benzylaminesulfonic acid associate with kringle 5 with similar affinities (Ka approximately 3.4 and 2.2 mM-1, respectively) while benzylamine binds about twice as tightly (Ka approximately 6.3 mM-1). The higher affinities toward both benzylamine and epsilon ACA indicate that a free carboxylate group is not, by itself, a main determinant of ligand-binding to kringle 5. The experiments also reveal a definite affinity for L-arginine methyl ester, L-lysine, and N alpha-acetyl-L-lysine methyl ester. It is suggested that, although weak (0.1 approximately less than Ka approximately less than 0.6 mM-1), these interactions could be of physiological relevance in the context of plasminogen binding to the fibrin clot. Ligand-induced shifts of kringle 5 proton resonances indicate that the Trp25, His33, Tyr50, Trp62, and Tyr72 (kringle numbering convention) side chains form or neighbor the kringle 5-binding site. Benzamidine-kringle 5 magnetization transfer (Overhauser) experiments verify a close proximity of the bound ligand to these aromatic groups. A model of the binding site is proposed in which the above residues interact closely with each other and define a lipophilic surface which is accessible to the free ligand.
利用300兆赫的1H核磁共振光谱研究了小分子与人纤溶酶原kringle 5结构域片段的结合情况。作为潜在配体进行测试的化合物包括L-精氨酸、L-赖氨酸以及一些大小相似但离子电荷构型不同的脂肪族和芳香族类似物。配体/kringle 5缔合常数（Ka）值是在22℃、pH 7.2条件下通过配体滴定实验获得的。L-精氨酸、Nα-乙酰-L-精氨酸和Nα-乙酰-L-精氨酸甲酯均未检测到与kringle 5的明显结合（Ka约小于0.05 mM-1）。相比之下，己胺或ε-氨基己酸（εACA）的结合更有利（Ka分别约为2.9和10.5 mM-1）。苯甲脒和对苄胺磺酸与kringle 5的缔合亲和力相似（Ka分别约为3.4和2.2 mM-1），而苄胺的结合紧密程度约为前者的两倍（Ka约为6.3 mM-1）。对苄胺和εACA的较高亲和力表明，游离羧基本身并非配体与kringle 5结合的主要决定因素。实验还揭示了对L-精氨酸甲酯、L-赖氨酸和Nα-乙酰-L-赖氨酸甲酯有一定亲和力。研究表明，尽管这些相互作用较弱（0.1约小于Ka约小于0.6 mM-1），但在纤溶酶原与纤维蛋白凝块结合的情况下可能具有生理相关性。配体诱导的kringle 5质子共振位移表明，Trp25、His33、Tyr50、Trp62和Tyr72（kringle编号惯例）侧链形成或邻近kringle 5结合位点。苯甲脒-kringle 5磁化转移（Overhauser）实验证实了结合的配体与这些芳香族基团紧密相邻。提出了一个结合位点模型，其中上述残基彼此紧密相互作用，形成一个可供游离配体接近的亲脂表面。
