Structure-function relationships of mitochondrial monoamine oxidase A and B: chimaeric 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, engineered by moving progressively the junction between the NH2- and the COOH-termini of each MAO form. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimaeras with increasing length of the NH2-terminus of MAO-A (up to position 256) showed a marked decrease in affinity towards substrates and inhibitors. Two sequences, spanning position 62 to 103 and 146 to 220, appeared of particular importance in putatively constituting the binding site of MAO-B. Conversely, the catalytic properties and specificity of MAO-A were insensitive to substitution of both the NH2-(up to position 112) and COOH-termini (from residue 395), but further modification of the central sequence of MAO-A was not compatible with activity. None of the engineered chimaeras showed a shift in substrate and inhibitor specificity. Investigation on MAO-B by site-directed mutagenesis revealed that His382 and Thr158 may represent residues relevant for MAO-B catalytic mechanism.
Cesura
A M
AM
Pharma Division, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
Gottowik
J
J
Lang
G
G
Malherbe
P
P
Da Prada
M
M
D019943:Amino Acid Substitution; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D006639:Histidine; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007668:Kidney; D007700:Kinetics; D008995:Monoamine Oxidase / Q000737:chemistry* / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D013912:Threonine; D014162:Transfection
D007527:Isoenzymes; D008996:Monoamine Oxidase Inhibitors; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D013912:Threonine; C063456:Ro 41-1049; D006639:Histidine; D008995:Monoamine Oxidase