Interactions of imidazoline ligands with the active site of purified monoamine oxidase A.

The two forms of monoamine oxidase, monoamine oxidase A and monoamine oxidase B, have been associated with imidazoline-binding sites (type 2). Imidazoline ligands saturate the imidazoline-binding sites at nanomolar concentrations, but inhibit monoamine oxidase activity only at micromolar concentrations, suggesting two different binding sites [Ozaita A, Olmos G, Boronat MA, Lizcano JM, Unzeta M & García-Sevilla JA (1997) Br J Pharmacol121, 901-912]. When purified human monoamine oxidase A was used to examine the interaction with the active site, inhibition by guanabenz, 2-(2-benzofuranyl)-2-imidazoline and idazoxan was competitive with kynuramine as substrate, giving K(i) values of 3 microM, 26 microM and 125 microM, respectively. Titration of monoamine oxidase A with imidazoline ligands induced spectral changes that were used to measure the binding affinities for guanabenz (19.3 +/- 3.9 microM) and 2-(2-benzofuranyl)-2-imidazoline (49 +/- 8 microM). Only one type of binding site was detected. Agmatine, a putative endogenous ligand for some imidazoline sites, reduced monoamine oxidase A under anaerobic conditions, indicating that it binds close to the flavin in the active site. Flexible docking studies revealed multiple orientations within the large active site, including orientations close to the flavin that would allow oxidation of agmatine.

Alteration in spectral properties on ligand binding reveals flexibility in monoamine oxidase.

Crystal structures have opened the door to understanding the mechanism and ligand specificities of MAO A and MAO B. We review here functional properties that suggest a flexibility in MAO that is likely to influence catalysis under different cellular conditions. The flexibility indicated by altered oxidation kinetics and a changed redox potential in the presence of a substrate was confirmed by circular dichroism spectroscopy. Circular dichroism also demonstrated alterations in the conformation of aromatic residues during reduction of MAO A and after covalent modification of the flavin. Visible spectra provide a convenient way to monitor ligand binding in the active site. Different groups near the flavin give different spectral changes. During reduction of MAO A, a distinct 412 nm peak appears after partial reduction. Recent work suggests that this may be a tyrosyl radical in equilibrium with the semiquinone of the flavin. Substrates prevent the appearance of the 412 nm peak but many inhibitors enhance it by preventing further reduction. We propose that steric effects in the active site could be the mechanism of this difference. Flexibility is also important for the transmission of the effects of modifying the surface thiols to the active site. Modification of multiple thiols results in inactivation but mutation of a single thiol, cysteine 374 in MAO A to alanine, decreased the catalytic potency (kcat/Km) by 30%. Thus, surface modification of MAO (for example, by oxidative stress) could reduce its activity.

Orientation of oxazolidinones in the active site of monoamine oxidase.

Oxazolidinone inhibitors of monoamine oxidase (MAO) and oxazolidinone antibacterials are two distinct classes of drug, often with linear structures and overlapping activities for some derivatives. By synthesizing novel dimerised derivatives with identical substitution of the two C-5 side chains, we have obtained experimental evidence for the orientation of oxazolidinones in the active site of MAO A. Two types of spectral changes, either increasing the absorbance at 510 nm or decreasing it at 495 nm depending on the group nearest to the flavin cofactor, were seen on ligand binding to MAO A. Side chain derivatives with amine substituents are very poor substrates so that it was possible to examine the spectral change due to binding of a substrate before reduction of the flavin occurred. Binding of these amino derivative substrates to MAO A induced a spectral change characterized by a strong decrease in absorbance at 495 nm. These substrates reduced the enzyme fully without any trace of a semiquinone intermediate. Only oxazolidinone inhibitors with a bromo-imidazole substituent increased the yield of semiquinone intermediate obtained during chemical reduction. In accord with the experimental data, results of docking experiments showed that binding of the oxazolidinone ring in the aromatic cage close to the flavin was favored and that the nitrogen of the derivatives that were substrates was within van der Waals distance of N-5 of the flavin.