Identification of a region important for human monoamine oxidase B substrate and inhibitor selectivity.
Monoamine oxidase (MAO) A and B are flavoenzymes that catalyze the oxidative deamination of biogenic and xenobiotic amines. To search for domains that confer substrate and inhibitor selectivities, two chimeric proteins were constructed and expressed in yeast. The kinetic constants and IC50 values were determined for these chimeric enzymes using MAO-A/B selective substrates and inhibitors. Replacement of MAO-A amino acids 161-375 with the corresponding region of MAO-B, termed AB(161-375)A, converted MAO-A catalytic properties to MAO-B like ones. The specificity constants (k(cat)/K(m))for the oxidation of beta-phenylethylamine (PEA) (1.6 x 10(5) s-1 M-1) and benzylamine (2.4 x 10(4) s-1 M-1) by AB (161-375)A were similar to wild-type MAO-B (PEA, 8 x 10(5)s(-1) M(-1); benzylamine, 4.9 x 10(4) s(-1) M(-1). Serotonin (5-HT), a preferred substrate for MAO-A, was not oxidized by AB(161-375)A or wild-type MAO-B. Furthermore, (AB161-375)A was more sensitive to the MAO-B specific inhibitor deprenyl (IC50 2.7 +/- 0.4 x 10(-8) M) than to the MAO-A specific inhibitor clorgyline (IC50 5.4 +/- 0.8 x 10(-7) M). However, the reciprocal chimera in which a MAO-B segment was replaced with the corresponding region of MAO-A, termed ++(+BA152-366B), lacked catalytic activity. The lack of catalytic activity was not due to aberrant expression but rather an inactive protein as demonstrated by Western blot analysis. These results demonstrate that MAO-B amino acids 152-366 contain a domain(s) that confers substrate and inhibitor selectivity.
Grimsby
J
J
University of Southern California, Department of Molecular Pharmacology and Toxicology, School of Pharmacy, Los Angeles, CA 90033, USA.
Zentner
M
M
Shih
J C
JC
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003010:Clorgyline / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae; D012642:Selegiline / Q000494:pharmacology
D007527:Isoenzymes; D008996:Monoamine Oxidase Inhibitors; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012642:Selegiline; D008995:Monoamine Oxidase; D003010:Clorgyline