Characterisation of neprilysin (EC 3.4.24.11) S₂′ subsite

Abstract

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 nonmutated and mutated enzymes with dipeptide inhibitors and hydrolysis of the substrate [Leu 5 , Arg 6 ]enkephalin in order to evaluate the contribution of the S 2 ' 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 P 2 ' residue of the substrate, (2) the S 2 ' subsite is vast and can accommodate bulky side chains, and (3) Arg-102 restricts access to the S 2 ' subsite to some side chains such as arginine.

A Three-dimensional Model of the Neprilysin 2 Active Site Based on the X-ray Structure of Neprilysin

“…Expression of rNEP2(s)-N567G in AtT20 cells and study of its catalytic activity using the model substrate showed that the K M of this mutant toward the fluorogenic substrate was significantly increased (p Յ 0.001) as compared with the wild type enzyme, whereas its k cat decreased by ϳ3-fold (p ϭ 0.0005), yielding an enzyme with a 43-fold decrease in its specificity constant (Table I). The significant effect of this mutation on the catalytic parameters of NEP2, on one hand, confirms the critical role of the Asn 567 residue in hydrolysis and in binding of the substrate as had been previously demonstrated for NEP (46) and, on the other hand, lends further credence to our proposed model of the NEP2 active site.…”

“…Indeed, the mutation of these residues in NEP (46) had previously shown a stronger interaction of Asn 542 than Arg 102 with the substrate. The 10-fold decrease in affinity of the rNEP2(s)-N567G mutant toward the model substrate confirms the involvement of this residue in substrate binding with this effect being furthermore comparable to a ϳ14-fold decrease in affinity observed for a NEP-N542G mutant (45)(46). Hydrolysis was also affected by the introduction of the N567G mutation within the NEP2 sequence, underlying the critical role of this residue in catalytic activity.…”

“…Of the various Arg 102 NEP mutants previously produced, an R102M mutant tested against various Phe-X dipeptides showed that their inhibitory potencies were generally decreased by this mutation compared with wild-type NEP (46), as observed here for NEP2 (Table II). The affinity of Phe-Gly toward NEP, the binding of which was of the most severely affected by the mutation of Arg 102 , decreased from the micromolar to millimolar range for wild-type and R102M NEP mutant, respectively, a difference in binding observed with NEP2 and thiorphan, which is constituted by a phenylalanine in PЈ 1 and a glycine residue in PЈ 2 (Table IV) (46). The loss of this active site arginine residue in both NEP and NEP2 (Arg 102 or Arg 131 , respectively) has an equivalent impact on the binding of PЈ 2 phenylalanine residues (5.5-versus 6-fold decrease in the binding of X-Phe to NEP and to NEP2, respectively), and the binding of enkephalins to NEP has also been shown to be compromised by the loss of Arg 102 in NEP (46,48) as demonstrated here for [Leu 5 ]enkephalin as well as for cholecystokinin 1-6 using the rNEP2(s)-R131M mutant.…”

“…The affinity of Phe-Gly toward NEP, the binding of which was of the most severely affected by the mutation of Arg 102 , decreased from the micromolar to millimolar range for wild-type and R102M NEP mutant, respectively, a difference in binding observed with NEP2 and thiorphan, which is constituted by a phenylalanine in PЈ 1 and a glycine residue in PЈ 2 (Table IV) (46). The loss of this active site arginine residue in both NEP and NEP2 (Arg 102 or Arg 131 , respectively) has an equivalent impact on the binding of PЈ 2 phenylalanine residues (5.5-versus 6-fold decrease in the binding of X-Phe to NEP and to NEP2, respectively), and the binding of enkephalins to NEP has also been shown to be compromised by the loss of Arg 102 in NEP (46,48) as demonstrated here for [Leu 5 ]enkephalin as well as for cholecystokinin 1-6 using the rNEP2(s)-R131M mutant. Although amidation of [Leu 5 ]enkephalin and the loss of the interaction between its free carboxyl group and Arg 131 decrease its binding to wild-type NEP2, the affinity of naturally amidated substrates was not significantly affected by the loss of Arg 131 (i.e.…”

The use of Fluorescence Resonance Energy Transfer (FRET) peptidesfor measurement of clinically important proteolytic enzymes

“…Although the enzyme has been first described as an endopeptidase (Kerr and Kenny 1974), in vitro studies have shown that NEP has better carboxydipeptidase than endopeptidase activity when the two situations of cleavage are possible (Malfroy and Schwartz 1982, Dion et al 1997.…”