Le Moual H, Devault A, Roques B P, Crine P, Boileau G
Département de Biochimie, Faculté de Médecine, Université de Montréal, Quebec, Canada.
J Biol Chem. 1991 Aug 25;266(24):15670-4.
Neutral endopeptidase (EC 3.424.11, NEP) is a membrane-bound zinc-metallopeptidase. The substrate specificity and catalytic activity of NEP resemble those of thermolysin, a bacterial zinc-metalloprotease. Comparison of the primary structure of both enzymes suggests that several amino acids present in the active site of thermolysin are also found in NEP. Using site-directed mutagenesis of the cDNA encoding the NEP sequence, we have already shown that His residues 583 and 587 are two of the three zinc ligands. In order to identify the third zinc ligand, we have substituted Val or Asp for Glu616 or Glu646. Val616 NEP showed the same kinetic parameters as the non-mutated NEP. In contrast, the mutant Val646 NEP was almost completely devoid of catalytic activity and unable to bind the tritiated inhibitor [3H]N-[2(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxypropyl]gl ycine, the binding of which is dependent on the presence of the zinc ion. Replacing Glu for Asp at position 646 conserved the negative charge, and the mutant enzyme exhibited the same Km value as the non-mutated enzyme, but kCat was decreased to less than 3% of the value of the non-mutated enzyme. When compared to the non-mutated enzyme Asp646 NEP showed a higher susceptibility to chelating agents, but bound the tritiated inhibitor with the same affinity. Taken together, these observations strongly suggest that Glu646 of NEP is the third zinc-coordinating residue and is equivalent to Glu166 in thermolysin.
中性内肽酶（EC 3.424.11，NEP）是一种膜结合锌金属肽酶。NEP的底物特异性和催化活性与嗜热菌蛋白酶（一种细菌锌金属蛋白酶）相似。两种酶一级结构的比较表明，嗜热菌蛋白酶活性位点中的几个氨基酸在NEP中也存在。通过对编码NEP序列的cDNA进行定点诱变，我们已经表明组氨酸残基583和587是三个锌配体中的两个。为了确定第三个锌配体，我们用缬氨酸或天冬氨酸取代了谷氨酸616或谷氨酸646。缬氨酸616 NEP显示出与未突变的NEP相同的动力学参数。相反，突变体缬氨酸646 NEP几乎完全没有催化活性，并且无法结合氚化抑制剂[3H]N-[2(R,S)-3-羟基氨基羰基-2-苄基-1-氧丙基]甘氨酸，其结合取决于锌离子的存在。在位置646处用天冬氨酸取代谷氨酸保留了负电荷，并且突变酶表现出与未突变酶相同的Km值，但kCat降低到未突变酶值的不到3%。与未突变的酶相比，天冬氨酸646 NEP对螯合剂的敏感性更高，但以相同的亲和力结合氚化抑制剂。综上所述，这些观察结果强烈表明NEP的谷氨酸646是第三个锌配位残基，并且相当于嗜热菌蛋白酶中的谷氨酸166。
