Dion N, Le Moual H, Crine P, Boileau G
Département de Biochimie, Faculté de Médecine, Université de Montréal, Canada.
FEBS Lett. 1993 Mar 8;318(3):301-4. doi: 10.1016/0014-5793(93)80533-z.
Neutral endopeptidase 24.11 (EC 3.4.24.11; NEP) is a membrane-bound Zn-metalloendopeptidase with a catalytic activity and a specificity very similar to that of thermolysin, a bacterial zinc-endoprotease. NEP can be inactivated by reaction with diethylpyrocarbonate, due to the modification of a histidine residue present in the active site of the enzyme. This histidine residue was proposed to be analogous to His231 in thermolysin, which is involved in the stabilization of the tetrahedral intermediate during the transition state. Using site-directed mutagenesis of the cDNA encoding rabbit NEP, we have created two mutants of NEP where His711 was replaced by either Gln or Phe (NEP-Gln711 and NEP-Phe711). Determination of kinetic parameters showed that both mutants had Km values very similar to that of the non-mutated enzyme but that their kcat values were 25-fold lower. The calculated difference in free energy needed to form the transition state complex was increased by 2.2 kcal/mol for both mutants. These observations strongly suggest that His711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.
中性内肽酶24.11（EC 3.4.24.11；NEP）是一种膜结合的锌金属内肽酶，其催化活性和特异性与嗜热菌蛋白酶（一种细菌锌内蛋白酶）非常相似。由于酶活性位点中存在的组氨酸残基发生修饰，NEP可通过与焦碳酸二乙酯反应而失活。该组氨酸残基被认为类似于嗜热菌蛋白酶中的His231，后者在过渡态期间参与四面体中间体的稳定。通过对编码兔NEP的cDNA进行定点诱变，我们构建了两种NEP突变体，其中His711被Gln或Phe取代（NEP-Gln711和NEP-Phe711）。动力学参数的测定表明，两种突变体的Km值与未突变酶的Km值非常相似，但其kcat值低25倍。两种突变体形成过渡态复合物所需的自由能计算差值增加了2.2千卡/摩尔。这些观察结果强烈表明，His711通过与四面体中间体的氧阴离子形成氢键参与过渡态的稳定。
