STRUCTURE-FUNCTION-RELATIONSHIPS OF MITOCHONDRIAL MONOAMINE-OXIDASE-AAND MONOAMINE-OXIDASE-B - CHIMERIC ENZYMES AND SITE-DIRECTED MUTAGENESIS STUDIES

To gain insight into the structure of monoamine oxidases (MAO) A and B , we investigated the properties of various chimaeric enzymes, enginee red by moving progressively the junction between the NH2- and the COOH -termini of each MAO form. Whereas exchange of the ADP-binding sequenc e did not modify the catalytic properties of either MAO isoforms, chim aeras with increasing length of the NH2-terminus of MAO-A (up to posit ion 256) showed a marked decrease in affinity towards substrates and i nhibitors. Two sequences, spanning position 62 to 103 and 146 to 220, appeared of particular importance in putatively constituting the bindi ng site of MAO-B. Conversely, the catalytic properties and specificity of MAO-A were insensitive to substitution of both the NH2- (up to pos ition 112) and COOH-termini (from residue 395), but further modificati on of the central sequence of MAO-A was not compatible with activity. None of the engineered chimaeras showed a shift in substrate and inhib itor specificity. Investigation on MAO-B by site-directed mutagenesis revealed that His382 and Thr158 may represent residues relevant for MA O-B catalytic mechanism.