Mutation in the substrate-binding site of aminopeptidase B confers new enzymatic properties

Pham, Viet-Laï; Gouzy-Darmon, Cécile; Pernier, Julien; Hanquez, Chantal; Hook, Vivian; Beinfeld, Margery C.; Nicolas, Pierre; Etchebest, Catherine; Foulon, Thierry; Cadel, Sandrine

doi:10.1016/j.biochi.2010.12.015

Cited by 10 publications

(9 citation statements)

References 51 publications

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“…This substitution shifted the Leishmania enzyme specificity towards that of cathepsin B. The importance of one amino acid residue for substrate specificity has also been shown for the representatives of several metallopeptidase families: aminopeptidase B (M1 family) (Pham et al, 2011), thimet oligopeptidase, and neurolysin (M3 family) (Oliveira et al, 2003;Kadonosono et al, 2008). In the M4 family (thermolysin-like proteases, Brought to you by | University of Connecticut Authenticated Download Date | 5/27/15 6:23 PM TLPs), the S1′ pocket has been demonstrated as a major determinant of the substrate specificity.…”

mentioning

confidence: 79%

Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III

Abramić

Karačić

Šemanjski

et al. 2015

Biological Chemistry

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Human dipeptidyl peptidase III (hDPP III) is a member of the M49 metallopeptidase family, which is involved in intracellular protein catabolism and oxidative stress response. To investigate the structural basis of hDPP III preference for diarginyl arylamide, using site-directed mutagenesis, we altered its S2 subsite to mimic the counterpart in yeast enzyme. Kinetic studies revealed that the single mutant D496G lost selectivity due to the increase of the Km value. The D496G, but not S504G, showed significantly decreased binding of peptides with N-terminal arginine, and of tynorphin. The results obtained identify Asp496 as an important determinant of human DPP III substrate specificity.

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mentioning

confidence: 79%

Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III

Abramić

Karačić

Šemanjski

et al. 2015

Biological Chemistry

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“…Moreover, based on the 3D structure of an LTA 4 H [E296Q] mutant in complex with an RAR substrate [12], a 3D Ap-B model with the RAR peptide docked in the active site was obtained ( Fig. 9) [16] that shows that the hydroxyl group of Y 229 does not interact directly with the substrate, but can contract a hydrogen bond with E 301 . This essential residue, located at the end of a b strand defined by residues of the GAMEN motif, interacts with the NH 3þ -terminus of the substrate (Fig.…”

Section: Tyrosine 229mentioning

confidence: 99%

“…The proximity between Y 414 and Y 409 is shown (1.84 Å distance). The RAR tripeptide has been docked by superposition of the Ap-B and LTA 4 H (pdb: 3b7t) structures with the DaliLite software of the Thornton group at the EMBL-EBI [16]. Images of structures were performed using the Visual Molecular dynamics (VMD) software.…”

Section: Tyrosine 229mentioning

confidence: 99%

“…Since there is no Ap-B structural data available, a 3D model was built based on the crystallographic structure of LTA 4 H [28]. Additionally, the cocrystallization of the LTA 4 H (E296Q) mutant with the R-A-R tripeptide substrate (pdb: 3B7T; 12) revealed important structural information that have allowed a better understanding of the enzymatic mechanism of Ap-B after docking of R-A-R ligand in the active site [16].…”

Section: Introductionmentioning

confidence: 99%

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The M1 family of vertebrate aminopeptidases: Role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B

et al. 2015

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Aminopeptidase B (Ap-B), a member of the M1 family of Zn(2+)-aminopeptidases, removes basic residues at the NH2-terminus of peptides and is involved in the in vivo proteolytic processing of miniglucagon and cholecystokinin-8. M1 enzymes hydrolyze numerous different peptides and are implicated in many physiological functions. As these enzymes have similar catalytic mechanisms, their respective substrate specificity and/or catalytic efficiency must be based on subtle structural differences at or near the catalytic site. This leads to the hypothesis that each primary structure contains a consensus structural template, strictly necessary for aminopeptidase activity, and a specific amino acid environment localized in or outside the catalytic pocket that finely tunes the substrate specificity and catalytic efficiency of each enzyme. A multiple sequence alignment of M1 peptidases from vertebrates allowed to identify conserved tyrosine amino acids, which are members of this catalytic backbone. In the present work, site-directed mutagenesis and 3D molecular modeling of Ap-B were used to specify the role of four fully (Y281, Y229, Y414, and Y441) and one partially (Y409) conserved residues. Tyrosine to phenylalanine mutations allowed confirming the influence of the hydroxyl groups on the enzyme activity. These groups are implicated in the reaction mechanism (Y414), in substrate specificity and/or catalytic efficiency (Y409), in stabilization of essential amino acids of the active site (Y229, Y409) and potentially in the maintenance of its structural integrity (Y281, Y441). The importance of hydrogen bonds is verified by the Y229H substitution, which preserves the enzyme activity. These data provide new insights into the catalytic mechanism of Ap-B in the M1 family of aminopeptidases.

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“…Simulations with or without the transition state analog inhibitor L-GluPO 3 H 2 revealed that the Arg368 residue stabilized the GAMEN motif by interacting directly with residues flanking this region (the pre-GAMEN loop). Several groups have shown the GAMEN motif to be crucial for gluzincin aminopeptidase activity, because this conserved motif is responsible of recognition of the free N-terminal part of substrates or inhibitors through the Glu residue of the motif [18,[32][33][34]. This interaction, which is initiated on formation of the Michaelis complex, affects catalysis by allowing optimal positioning of the substrate within the active site to trigger its hydrolysis [18,[32][33][34].…”

Section: Discussionmentioning

confidence: 99%

Structural insight into the catalytic mechanism and inhibitor binding of aminopeptidase A

Couvineau

Almeida

Leroux

et al. 2020

Biochemical Journal

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Aminopeptidase A (APA) is a membrane-bound monozinc aminopeptidase. In the brain, APA generates angiotensin III which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals. The oral administration of RB150 renamed firibastat by WHO, an APA inhibitor prodrug, targeting only the S1 subsite, decreases blood pressure in hypertensive patients from various ethnic origins. To identify new families of potent and selective APA inhibitors, we explored the organization of the APA active site, especially the S2’ subsite. By molecular modeling, docking, molecular dynamics simulations and site-directed mutagenesis, we revealed that Arg368 and Arg386, in the S2’ subsite of human APA established various types of interactions in major part with the P2’residue but also with the P1' residue of APA inhibitors, required for their nanomolar inhibitory potency. We also demonstrated an important role for Arg368 in APA catalysis, in maintaining the structural integrity of the GAMEN motif, a conserved sequence involved in exopeptidase specificity and optimal positioning of the substrate in monozinc aminopeptidases. This arginine together with the GAMEN motif are key players for the catalytic mechanism of these enzymes.

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Mutation in the substrate-binding site of aminopeptidase B confers new enzymatic properties

Cited by 10 publications

References 51 publications

Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III

Aspartate 496 from the subsite S2 drives specificity of human dipeptidyl peptidase III

The M1 family of vertebrate aminopeptidases: Role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B

Structural insight into the catalytic mechanism and inhibitor binding of aminopeptidase A

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