Le Moual H, Roques B P, Crine P, Boileau G
Département de Biochimie, Faculté de Médecine, Université de Montréal, Canada.
FEBS Lett. 1993 Jun 14;324(2):196-200. doi: 10.1016/0014-5793(93)81392-d.
Neutral endopeptidase (EC 3.4.24.11; NEP) is a membrane-bound zinc-metallopeptidase. The catalytic zinc ion is coordinated to three amino acid residues (His538, His587 and Glu646) and a water molecule. Here, we have systematically substituted potential metal-coordinating amino acid residues (His, Glu, Asp, Cys, Tyr, Ser) for each of the three zinc ligands of NEP using a recombinant polymerase chain reaction procedure. NEP mutants at positions 583 and 587 were devoid of catalytic activity. However, Glu587 NEP and Cys583 NEP were able to bind partially a tritiated inhibitor, the binding of which is dependent on the presence of the zinc atom. At position 646, the aspartate and cysteine mutants exhibited activity. For both mutants Km values were unaltered but kcat values were decreased by about 20-fold. Both mutants bound the tritiated inhibitor with Kd values similar to that of the wild-type enzyme. Our data suggest that neither histidine-583 nor -587 can be replaced by any other ligands. On the other hand, the glutamic acid at position 646 can be converted to an aspartic acid or a cysteine indicating the importance of a negative charge at this position.
中性内肽酶（EC 3.4.24.11；NEP）是一种膜结合锌金属肽酶。催化锌离子与三个氨基酸残基（His538、His587和Glu646）以及一个水分子配位。在此，我们使用重组聚合酶链反应程序，将潜在的金属配位氨基酸残基（His、Glu、Asp、Cys、Tyr、Ser）系统地取代NEP的三个锌配体中的每一个。位于583和587位的NEP突变体没有催化活性。然而，Glu587 NEP和Cys583 NEP能够部分结合一种氚标记的抑制剂，其结合依赖于锌原子的存在。在646位，天冬氨酸和半胱氨酸突变体表现出活性。对于这两个突变体，Km值未改变，但kcat值降低了约20倍。两个突变体结合氚标记抑制剂的Kd值与野生型酶相似。我们的数据表明，组氨酸-583和-587都不能被任何其他配体取代。另一方面，646位的谷氨酸可以转化为天冬氨酸或半胱氨酸，这表明该位置负电荷的重要性。
