Inhibition of alcohol dehydrogenase by bismuth

Abstract

Bismuth compounds have been widely used for the treatment of ulcers and Helicobacter pylori infection, and enzyme inhibition was thought to be crucial for bismuth anti-microbial activity. We have investigated the interaction of colloidal bismuth subcitrate (CBS) with alcohol dehydrogenase and our results demonstrate that bismuth can effectively inhibit the enzyme. Kinetic analysis revealed that CBS acted as a non-competitive inhibitor of yeast alcohol dehydrogenase. Both UV-vis and fluorescence data show that interaction of CBS with the enzyme exhibits biphasic processes. Bismuth can replace only half of Zn(II) from the enzyme (i.e., about one Zn(II) per monomer). Surprisingly, binding of CBS also induces the enzyme dissociation from its native form, tetramer into dimers. The inhibition of Bi(III) on the enzyme is probably due to its direct interference with the zinc sites. This study is likely to provide an insight into the mechanism of action of bismuth drugs.

Scheme 1

Sequence comparison of alcohol dehydrogenase from various sources, HLADH, horse liver alcohol dehydrogenase (gene code: M64864); HPADH, Helicobacter pylori alcohol dehydrogenase (gene code: JHP1429); YADH, yeast alcohol dehydrogenase (gene code: YOL086C). Those highlighted Cys and His residues are in either catalytic (light gray) or structural site (dark gray). (For a colour version of the figure see the online paper. Here light gray refers to yellow; dark gray refers to purple.)

Fig. 1

Lineweaver–Burk plots of the oxidation of ethanol catalyzed YADH, showing uninhibited and inhibited reactions. The assay system is comprised of 2.5 nM YADH and 1.5 mM NAD⁺ at 298 K, 20 mM Tris–HCl, pH 8.0. K_m for uninhibited reaction (■) was found to be 6.98 mM. The value is similar in the presence of 80 (▴) and 320 (♦) mol equiv of Bi(cit).

Fig. 2

(a) Dependence of absorption spectrum on time for a solution containing YADH (6 μM) and 40 mol equiv of Bi(cit) in 20 mM Tris–HCl at pH 8.0, 298 K. The broad band centered at 350 nm is indicative of formation of Bi(III)-S (thiolate) bonds. Reaction times from bottom to top: 0, 2, 4, 6, 15, 20, 30, 60 and 300 min. (b) Kinetics of the reaction of Bi(III) to YADH at 350 nm. The dependence of absorption spectrum on time for a solution containing YADH (6 μM) and 40 mol equiv of Bi(cit) in 20 mM Tris–HCl at pH 8.0, 298 K. Inset: semilog plot of the data and secondary plot of the fast step with a rate constant of 0.33 min⁻¹ and slow step with a rate constant of 5.5 × 10⁻² min⁻¹.

Fig. 3

(a) Dependence of fluorescence emission spectrum on time for a solution containing YADH (0.5 μM) and 40 mol equiv of Bi(cit) in 20 mM Tris–HCl at pH 8.0, 298 K with an excitation wavelength of 295 nm. The fluorescence emission intensity was significantly quenched after addition of Bi(cit). Reaction times from top to bottom: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 80, 100 and 120 min. (b) Dependence of fluorescence emission intensity at 340 nm on time. The assay solution contained YADH (0.5 μM) and 40 mol equiv of Bi(cit) in 20 mM Tris–HCl at pH 8.0, 298 K with excitation wavelength of 295 nm. Inset: semilog plot of the data and secondary plot of the fast step with a rate constant of 0.73 min⁻¹ and slow step with a rate constant of 7.7 × 10⁻² min⁻¹.

Fig. 4

FPLC profiles of YADH after incubation with Bi(cit) at different time. A solution of 10 μM YADH was incubated with 40 mol equiv of Bi(cit) in 0.1 M Tris–HCl, pH 8.0. At different time interval 200 μl mixture was separated by FPLC. From bottom to top, native YADH, incubation after 1, 40, 90 and 120 min, respectively. T – tetramer, D – dimer, M – monomer.

Fig. 5

Time dependence of peak areas obtained from FPLC chromatograms. YADH (10 μM) and 40 mol equiv of Bi(cit) were incubated in 20 mM Tris–HCl at pH 8.0, 298 K. At regular time interval, the solution was withdrawn and separated by FPLC. The tetramer peak area (■) gradually decreased while the dimer peak area (•) gradually increased. The monomer peak area (♦) remained almost unchanged.