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The catalytic and structural properties of divalent metal ion cofactor binding sites in the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. Co(II)-substituted DapE enzyme was 25% more active than the Zn(II)-loaded form of the enzyme. Interestingly, Mn(II) can activate DapE, but only to ∼20% of the Zn(II)-loaded enzyme. The order of the observed kcat values are Co(II) > Zn(II) > Cd(II) > Mn(II) >Ni(II) ∼ Cu(II) ∼ Mg(II). DapE was shown to only hydrolyze l,l-N-succinyl-diaminopimelic acid (l,l-SDAP) and was inactive toward d,l-, l,d-, and d,d-SDAP. DapE was also inactive toward several acetylated amino acids as well as d,l-succinyl aminopimelate, which differs from the natural substrate, l,l-SDAP, by the absence of the amine group on the amino acid side chain. These data imply that the carboxylate of the succinyl moiety and the amine form important interactions with the active site of DapE. The affinity of DapE for one versus two Zn(II) ions differs by nearly 2.2 × 103 times (Kd1 = 0.14 μM vs Kd2 = 300 μM). In addition, an Arrhenius plot was constructed from kcat values measured between 16 and 35 °C and was linear over this temperature range. The activation energy for [ZnZn(DapE)] was found to be 31 kJ/mol with the remaining thermodynamic parameters calculated at 25 °C being ΔG⧧ = 64 kJ/mol, ΔH⧧ = 28.5 kJ/mol, and ΔS⧧ = −119 J mol-1 K-1. Electronic absorption and EPR spectra of [Co_(DapE)] and [CoCo(DapE)] indicate that the first Co(II) binding site is five-coordinate, while the second site is octahedral. In addition, any spin−spin interaction between the two Co(II) ions in [CoCo(DapE)] is very weak. The kinetic and spectroscopic data presented herein suggest that the DapE from H. influenzae has similar divalent metal binding properties to the aminopeptidase from Aeromonas proteolytica (AAP), and the observed divalent metal ion binding properties are discussed with respect to their catalytic roles in SDAP hydrolysis.
Bienvenue, David L.; Gilner, Danuta M.; Davis, Ryan S.; Bennett, Brian; and Holz, Richard C., "Substrate Specificity, Metal Binding Properties, and Spectroscopic Characterization of the DapE-Encoded N-Succinyl-l,l-Diaminopimelic Acid Desuccinylase from Haemophilus influenzae" (2003). Physics Faculty Research and Publications. 39.
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