Tyrosine residues near the FAD binding site are critical for FAD binding and for the maintenance of the stable and active conformation of rat monoamine oxidase A.
Monoamine oxidase is a flavin-containing enzyme located at the mitochondrial outer membrane that catalyzes the oxidative deamination of amines. To investigate the role of tyrosine residues near the FAD-binding site, Cys-406, of monoamine oxidase A, the tyrosine residues at posiyions 402, 407, and 410 were indurdually replaced with alanine or phenylalanine and the effects of the mutations on catalytic activity, FAD binding, and enzyme structure were examined. Half or fewer of the mutant proteins incorporated FAD. The mutation of Tyr-407 to alanine led to an almost completely loss of catalytic activity for serotonin, PEA, tyramine, and tryptamine. A substantial decrease in the catalytic activity was also observed with the enzymes mutated at Tyr-402 and Tyr-410 to alanine, although the effect of the latter mutation was much less. All these mutants were sensitive to trypsin treatment of the purified enzyme, while the wild type enzyme was resistant to treatment. On the other hand, substitution of Tyr-402 or Tyr-407 with phenylalanine had little effect on these properties. Taken together, we conclude that tyrosine residues near Cys-406 may be form a pocket to facilitates FAD incorporation, the catalytic center, and a stable conformation, probably through interactions among the aromatic rings of the tyrosine residues and FAD.
Ma
Jichun
J
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
Ito
Akio
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts
D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine; D008995:Monoamine Oxidase; D003545:Cysteine