Phosphonium compounds as new and specific inhibitors of bovine serum amine oxidase

Abstract

TPP+ (tetraphenylphosphonium ion) and its analogues were found to act as powerful competitive inhibitors of BSAO (bovine serum amine oxidase). The binding of this new class of inhibitors to BSAO was characterized by kinetic measurements. TPP+ can bind to the BSAO active site by hydrophobic and by coulombian interactions. The binding probably occurs in the region of the 'cation-binding site'[Di Paolo, Scarpa, Corazza, Stevanato and Rigo (2002) Biophys. J. 83, 2231-2239]. Under physiological conditions, the association constant of TPP+ for this site is higher than 10(6) M(-1), the change of enthalpy being the main free-energy term controlling binding. Analysis of the relationships between substrate structure and extent of inhibition by TPP+ reveals some new molecular features of the BSAO active site.

Figure 1. Phosphonium compounds as competitive inhibitors of the SPM oxidase activity of BSAO

Lineweaver–Burk plots for the inhibition of BSAO by various inhibitors (A–C) are shown. All the measurements were performed in 25 mM Hepes/150 mM NaCl at pH 7.20 and 25 °C. The following concentrations for the various compounds were used: (A) PTP chloride at 0 (●), 0.1 (○) and 0.25 (□) μM; (B) TPP⁺ chloride at 0 (●), 0.25 (○), 0.5 (■), 1 (▲) and 10 (□) μM; and (C) TPAs chloride at 0 (●), 2 (○), 5 (△), 7.5 (□) and 10 (◆) μM. (D) Dixon plots obtained for the various inhibitors from the K_m values calculated from the double-reciprocal plots (A–C): O, TPAs chloride; ●, TPP⁺ chloride; ■, PTP chloride.

Figure 2. Inhibitory effect of TPP⁺ on BSAO activity using various substrates

A plot of the K_m,app/K_m,0 ratio against the TPP⁺ concentration is shown. All measurements were performed in 25 mM Hepes/150 mM NaCl, at pH 7.20 and 25 °C. The continuous curve was obtained by fitting experimental data to the Dixon equation for SPM, SPD and NONA, and to eqn (4) in the case of BUA and BZA. The following substrates were used: □, SPD; ●, SPM; ○, NONA; ▲, BZA; and △, BUA. Inset: magnification of the Figure at low TPP⁺ concentrations.

Scheme 1a

Scheme 1b

Figure 3. Dependence of BSAO–TPP⁺ K_b on pH

K_b values calculated at various pH values and at 25 °C are shown. SPD was used as substrate. The continuous line was obtained by non-linear least-squares fit of experimental data to eqn (2).

Figure 4. Arrhenius plot of the BSAO–TPP⁺ association constant versus temperature

Experiments were carried out in 25 mM Hepes/150 mM NaCl, at pH 7.2, using SPD as substrate.