Kinetic and spectroscopic characterization of the E134A- and E134D-altered dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae.

Glutamate-134 (E134) is proposed to act as the general acid/base during the hydrolysis reaction catalyzed by the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae. To date, no direct evidence has been reported for the role of E134 during catalytic turnover by DapE. In order to elucidate the catalytic role of E134, altered DapE enzymes were prepared in which E134 was substituted with an alanine and an aspartate residue. The Michaelis constant (K (m)) does not change upon substitution with aspartate but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.09%), and alanine (0%). Examination of the pH dependence of the kinetic constants k (cat) and K (m) for E134D-DapE revealed ionizations at pH 6.4, 7.4, and approximately 9.7. Isothermal titration calorimetry experiments revealed a significant weakening in metal K (d) values of E134D-DapE. D134 and A134 perturb the second divalent metal binding site significantly more than the first, but both altered enzymes can still bind two divalent metal ions. Structural perturbations of the dinuclear active site of DapE were also examined for two E134-substituted forms, namely E134D-DapE and E134A-DapE, by UV-vis and electron paramagnetic resonance (EPR) spectroscopy. UV-vis spectroscopy of Co(II)-substituted E134D-DapE and E134A-DapE did not reveal any significant changes in the electronic absorption spectra, suggesting that both Co(II) ions in E134D-DapE and E134A-DapE reside in distorted trigonal bipyramidal coordination geometries. EPR spectra of [Co_(E134D-DapE)] and [Co_(E1341A-DapE] are similar to those observed for [CoCo(DapE)] and somewhat similar to the spectrum of [Co(H(2)O)(6)](2+) which typically exhibit E/D values of approximately 0.1. Computer simulation returned an axial g-tensor with g ((x,y))=2.24 and E/D=0.07; g ( z ) was only poorly determined, but was estimated as 2.5-2.6. Upon the addition of a second Co(II) ion to [Co_(E134D-DapE)] and [Co_(E134A-DapE)], a broad axial signal was observed; however, no signals were observed with B (0)||B (1) ("parallel mode"). On the basis of these data, E134 is intrinsically involved in the hydrolysis reaction catalyzed by DapE and likely plays the role of a general acid and base.

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae is a dinuclear metallohydrolase.

The Zn K-edge extended X-ray absorption fine structure (EXAFS) spectra, of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae have been recorded in the presence of one or two equivalents of Zn(II) (i.e. [Zn_(DapE)] and [ZnZn(DapE)]). The Fourier transforms of the Zn EXAFS are dominated by a peak at ca. 2.0 A, which can be fit for both [Zn_(DapE)] and [ZnZn(DapE)], assuming ca. 5 (N,O) scatterers at 1.96 and 1.98 A, respectively. A second-shell feature at ca. 3.34 A appears in the [ZnZn(DapE)] EXAFS spectrum but is significantly diminished in [Zn_(DapE)]. These data show that DapE contains a dinuclear Zn(II) active site. Since no X-ray crystallographic data are available for any DapE enzyme, these data provide the first glimpse at the active site of DapE enzymes. In addition, the EXAFS data for DapE incubated with two competitive inhibitors, 2-carboxyethylphosphonic acid and 5-mercaptopentanoic acid, are also presented.

Substrate specificity, metal binding properties, and spectroscopic characterization of the DapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase from Haemophilus influenzae.

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 approximately 20% of the Zn(II)-loaded enzyme. The order of the observed k(cat) values are Co(II) > Zn(II) > Cd(II) > Mn(II) >Ni(II) approximately equal Cu(II) approximately equal 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 x 10(3) times (K(d1) = 0.14 microM vs K(d2) = 300 microM). In addition, an Arrhenius plot was constructed from k(cat) values measured between 16 and 35 degrees 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 degrees C being DeltaG(++) = 64 kJ/mol, DeltaH(++) = 28.5 kJ/mol, and DeltaS(++) = -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.

1,4-Bis[(1-methyl-1-phenylethyl)peroxymethyl]benzene.

The title compound, C(26)H(30)O(4), is one of the first alkyl bis-peroxides to be structurally characterized. The molecule lies on a centre of inversion and therefore the terminal phenyl rings are parallel. Although there are three aromatic rings in the molecule, the C-O-O-C torsion angle of 163.10 (10) degrees is close to the value found in Me(3)COOCMe(3).

Hydrolysis of thionopeptides by the aminopeptidase from Aeromonas proteolytica: insight into substrate binding.

A series of L-leucine aniline analogues were synthesized that contained either a carbonyl or thiocarbonyl as a part of the amide bond. Additionally, the para-position on the phenyl ring of several substrates was altered with various electron-withdrawing or donating groups. The kinetic constants K(m) and k(cat) were determined for the hydrolysis of each of these compounds in the presence of the aminopeptidase from Aeromonas proteolytica (AAP) containing either Zn(II) or Cd(II). The dizinc(II) form of AAP ([ZnZn(AAP)]) was able to cleave both carbonyl and thiocarbonyl containing peptide substrates with similar efficiency. However, the dicadmium(II) form of AAP ([CdCd(AAP)]) was unable to cleave any of the carbonyl-containing compounds tested but was able to cleave the thionopeptide substrates. This is consistent with the borderline hard/soft nature of Zn(II) vs Cd(II). The trends observed in the K(m) values suggest that the oxygen atom of the amide bond directly interacts with the dinuclear active site of AAP. Heterodimetallic forms of AAP that contained one atom of Zn(II) and one of Cd(II) (i.e., [CdZn(AAP)] and [ZnCd(AAP)]) were also prepared. The K(m) values for the thionopeptides substrates are the smallest when Cd(II) is in the first metal binding site, suggesting that substrate binds to the first metal binding site. 1-Phenyl-2-thiourea (PTU) and urea (PU) were also examined to determine the differences between thionopeptide and peptide binding to AAP. PTU and PU were found to be competitive inhibitors of AAP with inhibition constants of 0.24 and 4.6 mM, respectively. The electronic absorption and EPR spectra of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] were recorded in the absence and presence of both PU and PTU. Spectral changes were observed for PTU binding to [CoCo(AAP)] and [CoZn(AAP)] but not for [ZnCo(AAP)], while no spectral changes were observed for any of the Co(II)-substituted forms of AAP upon the addition of PU. These data indicate that carbonyl binding occurs only at the first metal binding site. In light of the data presented herein, the substrate binding step in the proposed mechanism of AAP catalyzed peptide hydrolysis can be further refined.