Separation of tryptophan pyrrolooxygenase into three molecular forms. A study of their substrate specificities using tryptophyl-containing peptides and proteins.
Tryptophan pyrrolooxygenase from wheat germ was separated into three molecular forms by microgranular DEAE-cellulose using a stepwise or a linear gradient elution procedure. In the first case molecular forms A and B were eluted with 10 mM Tris/HCl buffer (pH 7.4) and molecular form C was eluted with 50 mM KCl in the same buffer. The same separation could also be achieved with a linear KCl gradient (0-100 mM) in 10 mM Tris/HCl buffer (pH 7.4). The three molecular forms of tryptophan pyrrolooxygenase oxidized L-, D-, DL-Trp as well as many Trp derivatives with formation of N-formylkynurenyl derivatives. They also efficiently oxidized Trp-Phe, Trp-Tyr, Trp-Ala, Ala-Trp, Trp-Gly, Gly-Trp, Trp-Leu, Leu-Trp, Pro-Trp and Val-Trp, although the dipeptides were oxidized at different rates by the three molecular forms. A number of tryptophyl-containing tetra-, penta-, octa-, nona- and decapeptides were also oxidized. The oligopeptides which were known to have a helical conformation were better substrates than the smaller oligopeptides which were devoid of the conformational factor. The three molecular forms of tryptophan pyrrolooxygenase oxidized the tryptophyl residues of lysozyme, pepsin, chymotrypsin, trypsin and bovine serum albumin. It was found that molecular form A oxidized the more exposed (or hydrophilic) Trp residues of the proteins, while molecular form C also oxidized the Trp residues of a more hydrophobic nature. The three molecular forms were inhibited by chelating agents (alpha, alpha'-dipyridyl, EDTA and omicron-phenanthroline), although they differed in their sensitivities to these agents. Their optimum temperatures and inactivation rates at 65 degrees C was also different.
B
B
Camoretti-Mercado
R B
RB
Frydman
D002848:Chromatography, DEAE-Cellulose; D002918:Chymotrypsin / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology; D014364:Tryptophan / Q000378:metabolism*; D014366:Tryptophan Oxygenase / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D010455:Peptides; D011506:Proteins; D014364:Tryptophan; D014366:Tryptophan Oxygenase; C048647:peptide-tryptophan 2,3-dioxygenase; D002918:Chymotrypsin