Structurally Distinct Active Sites in the Copper(II)-Substituted Aminopeptidases from Aeromonas proteolytica and Escherichia coli
Format of Original
American Chemical Society
Journal of the American Chemical Society
Original Item ID
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||(63/65Cu) = 1.77 × 10-2 cm-1, whereas at pH 9.65 g|| = 2.245, g⊥ = 2.056, and A||(63/65Cu) = 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 B0 ⊥ B1 (perpendicular) mode EPR spectrum but were intense in the B0 || B1 (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||(63/65Cu) = 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) gz directions parallel to each other and at an angle of ∼17° to the inter-copper vector (ℋ = βB·gCuA·SCuA + βB·gCuB·SCuB + [S·A·I]CuA + [S·A·I]CuB + [SCuA·J·SCuB]; g||(CuA,CuB) = 2.218, g⊥(CuA,CuB) = 2.060; A||(CuA,CuB)(63/65Cu) = 1.59 × 10-2 cm-1, Jisotropic = 50 cm-1, rCu-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.
Bennett, Brian; Antholine, William E.; D'Souza, Ventris M.; Chen, Guanjing; Ustinyuk, Leila; and Holz, Richard C., "Structurally Distinct Active Sites in the Copper(II)-Substituted Aminopeptidases from Aeromonas proteolytica and Escherichia coli" (2002). Physics Faculty Research and Publications. 11.