Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin

Abstract

The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitive substrates for neprilysin (neutral endopeptidase) and thermolysin that are highly resistant to serine proteases

“…These substrates are hydrolyzed at the Ala-Phe and Ala-Leu bonds, respectively, conforming with the primary specificity of the enzyme, known to cleave internal peptide bonds of peptides on the amino side of hydrophobic amino acid residues [1,6,13]. The hydrophobic Phe or Leu residues of the substrates interact with the primary specificity site of the enzyme S'~, whereas the two Ala residues interact with the secondary sub-sites St and S 2 (according to the nomenclature of Schechter and Berger; [21]).…”

“…Neutral endopeptidase (EC 3.4.24.11) or neprilysin (NEP), an ectoenzyme present on the surface of many cell types [1][2][3], is a zinc metalloendopeptidase with a broad specificity. It hydrolyzes internal peptide bonds of peptides on the amino side of hydrophobic amino acid residues and in this respect it resembles the well-characterized bacterial zinc metalloproteinase thermolysin (EC 3.4.24.27) [4][5][6]. NEP has been implicated in the metabolism and regulation of a variety of biologically active peptides, e.g.…”

“…Most of the highly sensitive synthetic substrates for NEP in current use are amino protected tripeptides linked to a chromogenic or fluorogenic group at the carboxy terminus, with a Phe or Leu at the carboxy terminus serving as the major hydrophobic residue [6,[12][13][14]. These substrates are used in two-stage assays whereby an externally added aminopeptidase releases the chromophore or fluorophore subsequent to enzymatic cleavage of the substrate by NEE Here we report on the hydrolysis of four pairs of N-blocked tripeptide 4-nitroanilide *Corresponding author.…”

Determining Protease Substrate Selectivity and Inhibition by Label-Free Supramolecular Tandem Enzyme Assays

“…Analysis of the initial reaction rates at varying substrate concentrations (Figure 3b) yielded the characteristic proteolytic constants (kcat/KM) for the different peptide sequences (inset of Figure 3b and Table 1). 61 Note that our tandem assays allow kinetic measurements for unlabeled peptides, while previous assays were carried out with peptides carrying fluorescent labels such as 2-naphthylamide (2NA) 62 or dansyl. 63 The structural differences prevent a direct comparison of the absolute proteolytic constants.…”

Proteolytic cleavage of synthetic fragments of vesicle‐associated membrane protein, isoform‐2 by botulinum type B neurotoxin

“…The observed specificity of BoNT/B, however, clearly places these toxins into a distinct class of proteases. Previous studies on the substrate specificity of several neutral proteinases using model peptide substrates have shown that the rate of proteolysis is most influenced by the nature of the amino acid residues at the site of cleavage with residues at positions P,, P,, P',, P; (Schechter and Berger, 1967) having most influence on the reaction rate (Bouvier et al, 1990;Makinen et al, 1989;Morihara and Tsuzuki, 1970;Pozsgay et al, 1986). For all neutral proteases studied it has been possible to define short (< 10 amino acids) peptide substrates which are efficiently cleaved by the enzyme.…”