Regions of the molecule responsible for substrate specificity of monoamine oxidase A and B: a chimeric enzyme analysis.
To examine regions of the monoamine oxidase (MAO, EC 1.4.3.4) molecule responsible for substrate recognition, a series of these enzymes, in which discrete regions in one molecule were substituted by corresponding sequences of the other, were constructed from the cDNAs of rat liver MAO A and MAO B and were expressed in yeast, Saccharomyces cerevisiae. Substrate specificities of the original and chimeric enzymes were examined in terms of the maximum activity (Vmax) and the affinity (Km) for serotonin, beta-phenylethylamine (PEA), and benzylamine. Chimeric enzymes with the amino-terminal portion (about 220 residues) and the amino-terminal and middle portions (about 400 residues) of MAO A and MAO B, respectively, exhibited substantially the same Km values as those of the parent enzymes. Extension of the substitution in the middle portion of a chimeric enzyme to the second half of the amino-terminal portion resulted in conversion of the Km values for serotonin to those of the counterpart. Data on relative Vmax values of the chimeric enzymes for the three substrates revealed that the relative catalytic activities were mainly determined by the presence of the middle portion. We conclude from these observations that the region between about residues 120-220 and about residues 50-400 is responsible for determination of the substrate specificity of MAO A and MAO B, respectively, while the middle portion, of about residues 220-400, may relate to the relative catalytic activity towards substrates.
Tsugeno
Y
Y
Department of Chemistry, Kyushu University, Higashi-ku, Fukuoka.
Hirashiki
I
I
Ogata
F
F
Ito
A
A
D001483:Base Sequence; D001596:Benzylamines / Q000737:chemistry; D001665:Binding Sites; D004791:Enzyme Inhibitors; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000737:chemistry*; D010627:Phenethylamines / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012701:Serotonin / Q000737:chemistry; D013379:Substrate Specificity
D001596:Benzylamines; D004791:Enzyme Inhibitors; D010627:Phenethylamines; D011993:Recombinant Fusion Proteins; C029261:phenethylamine; D012701:Serotonin; C030796:benzylamine; D008995:Monoamine Oxidase