Catalytic and regulatory properties of native and chymotrypsin-treated pyridoxine-5-phosphate oxidase.
Brain pyridoxine-5-P oxidase is activated by the tryptophan metabolites 3-hydroxyanthranilate and 3-hydroxykynurenine. 3-Hydroxyanthranilate at concentrations of 0.03 mM relieves the inhibition elicited by accumulation of the substrate pyridoxine-5-P (Ki = 60 microM). The results of fluorometric measurements indicate that four molecules of 3-hydroxyanthranilate bind to the dimeric enzyme (56 kDa) with an association constant of 5.5 x 10(4) M-1. Differential spectral measurements failed to detect any direct interaction between the cofactor FMN and the effector 3-hydroxyanthranilate. These results are consistent with the hypothesis that the effector molecules bind to sites of the dimeric protein distinct from the cofactor site. Limited chymotrypsin digestion of pyridoxine-5-P oxidase yields catalytically active species that are no longer susceptible to activation by 3-hydroxykynurenine. A polypeptide of 16 kDa containing FMN and endowed with full catalytic activity was isolated by ion-exchange chromatography. It is postulated that the structural domain associated with catalytic activity composes approximately one-half of the molecular mass of pyridoxine-5-P oxidase (28 kDa), whereas the remaining portion of the macromolecule contains regulatory binding sites.
Kwon
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O
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Kwok
F
F
Churchich
J E
JE
D000818:Animals; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism* / Q000494:pharmacology; D005486:Flavin Mononucleotide / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011734:Pyridoxaminephosphate Oxidase / Q000378:metabolism*; D012756:Sheep; D014364:Tryptophan / Q000031:analogs & derivatives / Q000494:pharmacology
D010446:Peptide Fragments; D005486:Flavin Mononucleotide; D014364:Tryptophan; D011734:Pyridoxaminephosphate Oxidase; D002918:Chymotrypsin