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Spectroscopic and Thermodynamic Characterization of the E151D and E151A Altered Leucine Aminopeptidases from Aeromonas proteolytica
Inorganic Chemistry
  • Krzysztof P. Bzymek, Utah State University
  • Sabina I. Swierczek, Utah State University
  • Brian Bennett, Marquette University
  • Richard C. Holz, Marquette University
Document Type
Format of Original
7 p.
Publication Date
American Chemical Society
Original Item ID
doi: 10.1021/ic051034g
Previous kinetic characterization of the glutamate 151 (E151)-substituted forms of the leucine aminopeptidase from Aeromonas proteolytica (Vibrio proteolyticus; AAP) has provided critical evidence that this residue functions as the general acid/base. The close proximity of similar glutamate residues to the bridging water/hydroxide of the dinuclear active sites of metalloenzymes (2.80 and 3.94 Å in carboxypeptidase G2 and 3.30 and 3.63 Å in AAP), suggests it may also be involved in stabilizing the active-site metal ions. Therefore, the structural perturbations of the dinuclear active site of AAP were examined for two E151-substituted forms, namely E151D-AAP and E151A-AAP, by UV−vis and electron paramagnetic resonance (EPR) spectroscopy. UV−vis spectroscopy of Co(II)-substituted E151A-AAP did not reveal any significant changes in the electronic absorption spectra. However UV−vis spectra of mono- and dicobalt(II) E151D-AAP exhibited a lower molecular absorptivity compared to AAP (23 and 43 M-1 cm-1 vs. 56 and 109 M-1 cm-1 for E151D-AAP and AAP, respectively) suggesting both Co(II) ions reside in distorted octahedral coordination geometry in E151D-AAP. EPR spectra of [Co_(E151D-AAP)], [ZnCo(E151D-AAP)], and [(CoCo(E151D-AAP)] were identical, with g⊥ = 2.35, g∥ = 2.19, and E/D = 0.19, similar to [CoCo(AAP)]. On the other hand, the EPR spectrum of [Co_(E151A-AAP)] was best simulated assuming the presence of two species with (i) gx,y = 2.509, gz = 2.19, E/D = 0.19, A = 0.0069 cm-1 and (ii) gx,y = 2.565, gz = 2.19, E/D = 0.20, A = 0.0082 cm-1 indicative of a five- or six-coordinate species. Isothermal titration calorimetry experiments revealed a large decrease in Zn(II) affinities, with Kd values elevated by factors of ∼850 and ∼24 000 for the first metal binding events of E151D- and E151A-AAP, respectively. The combination of these data indicates that E151 serves to stabilize the dinuclear active site of AAP.

Inorganic Chemistry, Vol. 44, No.23 (November 2005): 8574-8580. DOI.

Brian Bennett was affiliated with Medical College of Wisconsin at the time of publication.

Richard Holz was affiliated with Utah State University at the time of publication.

Citation Information
Krzysztof P. Bzymek, Sabina I. Swierczek, Brian Bennett and Richard C. Holz. "Spectroscopic and Thermodynamic Characterization of the E151D and E151A Altered Leucine Aminopeptidases from Aeromonas proteolytica" Inorganic Chemistry (2005) ISSN: 0020-1669
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