Active Site Rearrangement of the 2-Hydrazinopyridine Adduct in Escherichia coli Amine Oxidase to an Azo Copper(II) Chelate Form:  A Key Role for Tyrosine 369 in Controlling the Mobility of the TPQ−2HP Adduct

Abstract

Adduct I (lambda(max) at approximately 430 nm) formed in the reaction of 2-hydrazinopyridine (2HP) and the TPQ cofactor of wild-type Escherichia coli copper amine oxidase (WT-ECAO) is stable at neutral pH, 25 degrees C, but slowly converts to another spectroscopically distinct species with a lambda(max) at approximately 530 nm (adduct II) at pH 9.1. The conversion was accelerated either by incubation of the reaction mixture at 60 degrees C or by increasing the pH (>13). The active site base mutant forms of ECAO (D383N and D383E) showed spectral changes similar to WT when incubated at 60 degrees C. By contrast, in the Y369F mutant adduct I was not stable at pH 7, 25 degrees C, and gradually converted to adduct II, and this rate of conversion was faster at pH 9. To identify the nature of adduct II, we have studied the effects of pH and divalent cations on the UV-vis and resonance Raman spectroscopic properties of the model compound of adduct I (2). Strikingly, it was found that addition of Cu2+ to 2 at pH 7 gave a product (3) that exhibited almost identical spectroscopic signatures to adduct II. The X-ray crystal structure of 3 shows that it is the copper-coordinated form of 2, where the +2 charge of copper is neutralized by a double deprotonation of 2. These results led to the proposal that adduct II in the enzyme is TPQ-2HP that has migrated onto the active site Cu2+. The X-ray crystal structure of Y369F adduct II confirmed this assignment. Resonance Raman and EPR spectroscopy showed that adduct II in WT-ECAO is identical to that seen in Y369F. This study clearly demonstrates that the hydrogen-bonding interaction between O4 of TPQ and the conserved Tyr (Y369) is important in controlling the position and orientation of TPQ in the catalytic cycle, including optimal orientation for reactivity with substrate amines.

Human copper-dependent amine oxidases

“…Despite these important differences between AOC3 and other CAOs, the active site configuration of the 2-HP-inhibited form of rAOC3 is very similar to a previously characterized 2-HP-inhibited form of a CAO from E. coli (31,32), and confirmed that Asp386 is the active site base for AOC3 and that the pyridine ring of the 2-HP is involved in π-stacking interactions with Tyr (Tyr384). This Tyr residue is also conserved in AOC1 (Tyr371) and AOC2 (Tyr378) (43,59).…”

A new crystal form of human vascular adhesion protein 1

“…In chains A, B, D, E and G TPQ O4 sits at a distance of 3.1-3.5 Å from the hydroxyl group of Tyr372, a conserved residue in all known CAO structures that is believed to stabilize an 'off-copper' TPQ, and TPQ O5 is between 2.9 and 3.4 Å away from the carboxylate group of the catalytic base Asp386. This arrangement has been observed in most other CAOs in which the TPQ is 'offcopper' (Wilce et al, 1997;Li et al, 1998;Lunelli et al, 2005;Mure et al, 2005). However, in monomers C and F the distance from TPQ O4 to Tyr372 is longer (4.4 and 4.9 Å , respectively).…”

Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli

“… 58 Upon reaction of E573Q and I342F/E573Q with molar equivalents of 2HP, the azo form of adduct I and adduct II 70 were immediately observed as the UV–vis spectra show two peaks at 416 and 520 nm ( Figure 4 ). 59 A full conversion from the azo form to adduct II was observed with prolonged incubation at room temperature in I342F/E573Q. 59 By contrast, for WTECAO and I342F, only the hydrazone form of adduct I was detected.…”

“… 59 A full conversion from the azo form to adduct II was observed with prolonged incubation at room temperature in I342F/E573Q. 59 By contrast, for WTECAO and I342F, only the hydrazone form of adduct I was detected. No spectral change was observed for Y466F as expected ( Figure 4 ).…”