Inhibition of carboxypeptidase A by excess zinc: analysis of the structural determinants by X‐ray crystallography

Gomez-Ortiz, Mariola; Gomis-Rüth, F.X.; Huber, Robert; Avilés, Francesc X.

doi:10.1016/s0014-5793(96)01412-3

Cited by 83 publications

(51 citation statements)

References 32 publications

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“…3) is comparable to clusters observed in the active sites of zinc proteases similarly inhibited by excess zinc (31,32). The catalytic Zn A 2ϩ ion is more deeply situated in the active site than the inhibitory Zn B 2ϩ ion and is coordinated by H79 of helix A, H238 and D242 of helix AЈ, and a solvent molecule with tetrahedral geometry.…”

Section: Resultssupporting

confidence: 58%

“…3). X-ray crystal structures of the zinc proteases thermolysin and carboxypeptidase A reveal that inhibitory zinc ions interact with conserved glutamate residues E166 and E270, respectively (31,32). These residues serve as general bases in the corresponding peptidase reactions (33,34), and by analogy we propose that E78 of LpxC serves as a general base in the deacetylase reaction (Fig.…”

Section: Resultsmentioning

confidence: 87%

See 1 more Smart Citation

Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis

Whittington

Rusche

Shin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

155

228

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The outer leaflet of the outer membrane of the Gram-negative bacterium serves as a permeability barrier and is composed of lipopolysaccharide, also known as endotoxin. The membrane anchor of lipopolysaccharide is lipid A, the biosynthesis of which is essential for cell viability. The first committed step in lipid A biosynthesis is catalyzed by UDP-(3-O-(R-3-hydroxymyristoyl))-Nacetylglucosamine deacetylase (LpxC), a zinc-dependent deacetylase. Here we report the crystal structure of LpxC from Aquifex aeolicus, which reveals a new ␣؉␤ fold reflecting primordial gene duplication and fusion, as well as a new zinc-binding motif. The catalytic zinc ion resides at the base of an active-site cleft and adjacent to a hydrophobic tunnel occupied by a fatty acid. This tunnel accounts for the specificity of LpxC toward substrates and inhibitors bearing appropriately positioned 3-O-fatty acid substituents. Notably, simple inhibitors designed to target interactions in the hydrophobic tunnel bind with micromolar affinity, thereby representing a step toward the structure-based design of a potent, broad-spectrum antibacterial drug.

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Section: Resultssupporting

confidence: 58%

Section: Resultsmentioning

confidence: 87%

Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis

Whittington

Rusche

Shin

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

155

228

View full text Add to dashboard Cite

show abstract

“…In addition, X-ray cyrstallography studies show that a second zinc binds to the enzyme active site that may perturb substrate catalysis [20]. These interesting studies, however, differ from zinc inhibition of AP-B shown in this report, since zinc shows mixed inhibition kinetics for AP-B (instead of zinc competitive inhibition of CPA).…”

Section: Discussioncontrasting

confidence: 55%

Zinc regulation of aminopeptidase B involved in neuropeptide production

Hwang

Hook

2008

FEBS Letters

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Aminopeptidase B (AP-B) is a metallopeptidase that removes basic residues from the N-termini of neuropeptide substrates in secretory vesicles. This study assessed zinc regulation of AP-B activity, since secretory vesicles contain endogenous zinc. AP-B was inhibited by zinc at concentrations typically present in secretory vesicles. Zinc effects were dependent on concentration, incubation time, and the molar ratio of zinc to enzyme. AP-B activity was recovered upon removal of zinc. AP-B with zinc became susceptible to degradation by trypsin, suggesting that zinc alters enzyme conformation. Zinc regulation demonstrates the metallopeptidase property of AP-B.

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“…This is one striking difference between the active site here and those in other binuclear zinc hydrolases such as the ␣␤ members, where the water/hydroxide ion is almost symmetrically located between the two metal ions (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Therefore, the inhibitory metal ion at the ␣ site may hold the active site water tightly and thus perturb the stereochemical arrangements required for enzyme catalysis as it does in bovine carboxypeptidase A (38). The much stronger inhibition by Zn 2ϩ and Cd 2ϩ than Cu 2ϩ may be due to Asp 366 , an important residue for the proton shuttle, and the active site water molecule being closer to the ␣ 1 subsite than the ␣ 2 subsite (2.61 Å).…”

Section: Resultsmentioning

confidence: 89%

“…Inhibition by Excess Zinc Ion in Other Metalloenzymes-The crystal structures of thermolysin and carboxypeptidase A, main representatives of two large superfamilies, endo-and exoproteases, in the presence of excess zinc also show second inhibitory zinc-binding sites at the active site (38,39). Both inhibitory zinc ions are also located in close proximity to the essential zinc (3.2 and 3.3 Å).…”

Section: Resultsmentioning

confidence: 99%

The Functional Role of the Binuclear Metal Center in d-Aminoacylase

Lai¹,

Chou²,

Ting³

et al. 2004

Journal of Biological Chemistry

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Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: ␣␤-binuclear, ␣-mononuclear, ␤-mononuclear, and metal-independent subsets. The D-aminoacylase from Alcaligenes faecalis DA1 belongs to the ␤-mononuclear subset due to the fact that the catalytically essential , revealing that elimination of the ␤ site changes the coordination geometry of the ␣ ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the Daminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the ␤ site, but inhibited by the subsequent binding of the second ion at the ␣ site.D-Aminoacylase (EC 3.5.1.81) is an attractive candidate for production of D-amino acids through its catalysis of the deacetylation of N-acetyl-D-amino acids. Since D-amino acids are intermediates in the preparation of antibiotics, pesticides, and bioactive peptides, D-aminoacylase has commercial importance for the optical resolution of N-acetyl-DL-amino acids (1-2). The crystal structure of the 483-residue D-aminoacylase from Alcaligenes faecalis DA1 revealed that the enzyme is comprised of a small ␤ barrel and a catalytic TIM barrel with a unique 63-residue insertion (3). The structural similarity and the conserved metal ligands of four histidines and one aspartate suggest that D-aminoacylase belongs to the TIM barrel metal-dependent hydrolase superfamily. This superfamily includes a variety of enzymes with highly diverse substrates and has been coined an "evolutionary treasure" (4).To date, crystal structures of 14 members in the superfamily have been determined. As more structures are solved, more divergences in the metal centers and in the ␤-strand packing of the TIM barrel are observed (Fig. 1). Based on the binding site(s) of the catalytically essential metal ion(s), we classify these members into four types: ␣␤-binuclear (␣␤), ␣-mononuclear (␣), ␤-mononuclear (␤), and metal-independent subsets. Phosphotriesterase (homology protein), urease, dihydroorotase, renal dipeptidase, isoaspartyl didpeptidase, and dihydropyrimidinase comprise the ␣␤ subset (5-15). Murine adenosine deaminase and Escherichia coli cytosine deaminase belong to the ␣ subset (16, 17). D-Aminoacylase belongs to the ␤ subset due to the essential Zn 2ϩ binding tightly at the ␤ site, even though it possesses a weak ␣ binding site (3). In contrast, the activity assay and the crystal structure of uronate isomerase show that this enzyme does not display any specific metal requirement and thus belongs to the metal-independent subset (18,19).The functional requirement for the different metal centers is still unclear. Interestingly, Bacillus subtilis N-acetylglucosamine-6-phosphate deacetylase ...

show abstract

Inhibition of carboxypeptidase A by excess zinc: analysis of the structural determinants by X‐ray crystallography

Cited by 83 publications

References 32 publications

Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis

Crystal structure of LpxC, a zinc-dependent deacetylase essential for endotoxin biosynthesis

Zinc regulation of aminopeptidase B involved in neuropeptide production

The Functional Role of the Binuclear Metal Center in d-Aminoacylase

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