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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 His231in 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 His711was replaced by either Gln or Phe (NEP‐Gln711and NEP‐Phe711). Determination of kinetic parameters showed that both mutants had Kmvalues very similar to that of the non‐mutated enzyme but that their kcatvalues 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 His711is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.

Direct comparison of the primary structure of neutral endopeptidase (NEP, EC 3.4.24.11) with that of thermolysin, a bacterial metalloendopeptidase with a similar specificity, has revealed very few similarities between the two sequences, except for two conserved short segments. In thermolysin, these segments contain several of the residues involved in catalysis, including two zinc coordinating histidines (His‐142 and His‐146) and a third histidine (His‐231) involved in stabilizing the transition state through hydrogen bonding. The role of the corresponding histidines in NEP (His‐583, His‐587 and His‐637) was explored by site‐directed mutagenesis of NEP cDNA and expression of the mutated cDNA in COS‐1 cells. Substitution of either His‐583 or His‐587 of NEP for Phe completely abolished the activity and Zn‐directed inhibitor recognition of the recombinant enzyme, suggesting that these residues play a role similar to His‐ 142 and His‐146 of thermolysin as zinc ligands. In contrast, substitution of His‐637 for a phenylalanine residue was without effect on enzyme activity.

Endopeptidase-24.18 (endopeptidase-2, EC 3.4.24.18, E-24.18) is a Zn-ectoenzyme of rat renal and intestinal microvillar membranes exhibiting an oligometric structure, α 2-β 2. The primary structure of the α-subunit of E-24.18 has been defined by molecular cloning and its expression mapped in rat kidney by in situ hybridization. A 2.9-kb cDNA coding for the α-subunit was isolated and sequenced. It had an open reading frame of 2.244 base pairs coding for a type I membrane protein of 748 amino acids. The deduced amino acid sequence showed 87% identity with that of meprin A, a mouse metallo-endopeptidase, sharing common properties with the rat enzyme, and 85% identity with the human intestinal enzyme, ‘PABA-peptide hydrolase’. Northern blot analysis revealed the α-subunit to be encoded by a single mRNA species of 3.2-kb. In situ hybridization performed on rat kidney showed a co-localization of E-24.18 with endopeptidase-24.11 in proximal tubules of juxtamedullary nephrons, suggesting that the two enzymes have similar or complementary physiological functions in kidney.

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 (His 538, Hi 587and Glu 646) 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, Glu 587NEP and Cys 583NEP 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 Kmvalues were unaltered but Kcatvalues were decreased by about 20-fold. Both mutants bound the tritiated inhibitor with Kdvalues 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.

Endopeptidase‐24.18 (EC 3.4.24.18, E‐24.18) is an oligomeric Zn‐ectoenzyme. The α and β submits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C‐terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E‐24.18 α subunit and to test the functionality of the astacin‐like domain in the α subunit when expressed alone, COS‐1 cells were transfected with a cloned cDNA for rat α subunit. Despite the presence of its putative transmembrane domain, the α subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the α subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu‐157 in the active site by Val. Taken together our results suggest that the α subunit of Endopeptidase‐24.18 contains a latent astacin‐like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine.

Neprilysin is a neutral peptidase that cleaves small peptide substrates on the amino‐side of hydrophobic amino acid residues. In the present study, we have used inhibition of non‐mutated and mutated enzymes with dipeptide inhibitors and hydrolysis of the substrate [Leu5, Arg6]enkephalin in order to evaluate the contribution of the S2′ subsite to substrate and inhibitor binding. Our results suggest that (1) Arg‐102 and Asn‐542 provide major contributions to the interaction of the enzyme with the P2′ residue of the substrate, (2) the S2′ subsite is vast and can accommodate bulky side chains, and (3) Arg‐102 restricts access to the S2′ subsite to some side chains such as arginine.