Spectroscopic and thermodynamic characterization of the E151D and E151A altered leucine aminopeptidases from Aeromonas proteolytica
Spectroscopic and thermodynamic characterization of the E151D and E151A altered leucine aminopeptidases from Aeromonas proteolytica
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DOI：
10.1021/ic051034g
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发表时间：
2005-11-14
影响因子：
4.6
通讯作者：
Holz, RC
中科院分区：
文献类型：
--
作者：
Bzymek, KP;Swierczek, SI;Holz, RC
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 angstrom in carboxypeptidase G(2) and 3.30 and 3.63 angstrom 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 E151 D-AAP and E151 A-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(perpendicular to) = 2.35, g(parallel to) = 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) g(x,y) = 2.509, g(z) = 2.19, E/D = 0.19, A = 0.0069 cm(-1) and (ii) g(x,y) = 2.565, g(z) = 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 K-d values elevated by factors of similar to 850 and similar to 24 000 for the first metal binding events of E151D- and E151 A-AAP, respectively. The combination of these data indicates that E151 serves to stabilize the dinuclear active site of AAP.
