Structurally Distinct Active Sites in the Copper(II)-Substituted Aminopeptidases from Aeromonas proteolytica and Escherichia coli

Abstract

The aminopeptidase from Aeromonas proteolytica (AAP) was titrated with copper, which bound sequentially at two distinct sites. Both the mono-and disubstituted forms of AAP exhibited catalytic hyperactivity relative to the native dizinc enzyme. Monosubstituted AAP exhibited an axial Cu(II) EPR spectrum with slight pH dependence: at pH 6.0 g || =2.249, g ⊥ = 2.055, and A || ( 63/65 Cu) = 1.77 × 10 −2 cm −1 , whereas at pH 9.65 g || = 2.245, g ⊥ = 2.056, and A || ( 63/65 Cu) = 1.77 × 10 −2 cm −1 . These data indicate oxygen and nitrogen ligation of Cu. AAP further substituted with copper exhibited a complex signal with features around g ~2 and 4. The features at g ~4 were relatively weak in the B 0 ⊥ B 1 (perpendicular) mode EPR spectrum but were intense in the B 0 || B 1 (parallel) mode spectrum. The g ~2 region of the perpendicular mode spectrum exhibited two components, one corresponding to mononuclear Cu(II) with g || = 2.218, g ⊥ = 2.023, and A || ( 63/65 Cu) = 1.55 × 10 −2 cm −1 and likely due to adventitious binding of Cu(II) to a site distant from the active site. Excellent simulations were obtained for the second component of the spectrum assuming that two Cu(II) ions experience dipolar coupling corresponding to an inter-copper distance of 5 Å with the two Cu(II) g z directions parallel to each other and at an angle of ~17° to the inter-copper vector (ℋ= βB·g CuA ·S CuA + βB·g CuB ·S CuB + [S·A·I] CuA + [S·A·I] CuB + [S CuA ·J·S CuB ]; g ||(CuA,CuB) = 2.218, g ⊥(CuA,CuB) = 2.060; A ||(CuA,CuB) ( 63/65 Cu) = 1.59 × 10 −2 cm −1 , J isotropic = 50 cm −1 , r Cu-Cu = 4.93 Å, and χ=17°). The exchange coupling between the two copper ions was found to be ferromagnetic as the signals exhibited Curie law temperature dependence. The Cu-Cu distance of ~5 Å indicated by EPR was significantly higher than the inter-zinc distance of 3.5 Å in the native enzyme, and the dicopper species therefore represents a novel dinuclear site capable of catalysis of hydrolysis. In contrast to AAP, the related methionyl aminopeptidase from Escherichia coli (EcMetAP) was found to bind only one Cu(II) ion despite possessing a dinuclear binding site motif. A further difference was the marked pH dependence of the signal in EcMetAP, suggestive of a change in ligation. The structural motifs of these two Cu(II)-substituted aminopeptidases provide important insight into the observed catalytic activity.

Role of the Zn1and Zn2sites in Metallo-β-lactamase L1

“…The second species exhibited no sharp resonances and was due to an axial species similar to that observed from Co(II) in L1 in earlier work20. Based on previous reports, these signals are consistent with an equilibrium of Co(II)-H 2 O and Co(II)-OH30-32. More typically, 1Co-L1 contained 0.9 − 1.0 eq Co(II), and the spectrum (Figure 2B) became less well-resolved, with only inflection points to suggest the presence of the distinct species observed at lower Co(II) complement.…”

Interactions between H-bonded [CuII3(μ3-OH)] triangles; a combined magnetic susceptibility and EPR study

“…reported for the much simpler case of Cu dimers. [62][63][64] Indeed, in the parallel-mode spectrum one characteristic derivativelike signal is observed at a position corresponding to the assumed forbidden transition. Fig.…”

Multi-Technique Characterization of Poly-L-lysine Dendrigrafts-Cu(II) Complexes for Biocatalysis

“…Different methods were tested for the computation of the low temperature spectra: (a) the CU23 program kindly provided by Prof. Romanelli, University of Florence, Italy; (b) the Bruker's WIN‐EPR SimFonia Software Version 1.25; (c) the method reported by Bennett et al; (d) the program EasySpin 4.5.1, using MATLAB 7.5; (e) the procedure by Budil et al for computing nitroxide radical spectra, which may be successfully applied to the simulation of Cu(II) spectra too. This last program was also used to compute the spectra at 298 K, thus providing information about the mobility of the Cu‐complex (the correlation time for the diffusion rotational motion of the complexed Cu(II) ions, τ; accuracy of ± 0.05 ns), which is related to the flexibility of DGL structure in the region where Cu 2+ is located.…”