Kinetic evidence that His‐711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state

Abstract 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 His 231 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 His 711 was replaced by either Gln or Phe (NEP-Gln 711 and NEP-Phe 711 ). Determination of kinetic parameters showed that both mutants had K m values very similar to that of the non-mutated enzyme but that their k cat 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 His 711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.