Isolation, crystallization, and properties of indolyl-3-alkane alpha-hydroxylase. A novel tryptophan-metabolizing enzyme.
Indolyl-3-alkane alpha-hydroxylase, a novel tryptophan-metabolizing enzyme, was prepared in crystalline form from soil isolate organism Pseudomonas XA. Emission spectroscopy and atomic absorption analyses of purified enzyme revealed the presence of iron (0.8 mol/mol of protein), and a number of observations supported the presence of heme prosthetic group (1.1 mol/mol of protein). The S20,w value of indolyl-3-alkane alpha-hydroxylase is 10.2 S, and the molecular weight by sedimentation equilibrium ultracentrifugation is 250,000. The E1%280 of the enzyme is 21, and the isoelectric point by isoelectric focusing on ampholine polyacrylamide gel plates is 4.8. The enzyme catalyzes hydroxylation on the side chain of a variety of 3-substituted indole compounds, including certain tryptophan-containing oligopeptides. The reaction product from tryptamine was identified by proton nuclear magnetic resonance and gas chromatography/mass spectroscopy analyses. While the indole ring remained intact, hydroxylation occurred at the side chain carbon adjacent to the ring. Nuclear magnetic resonance studies indicated that hydroxylation always took place at the same position when the substrate was tryptophan methyl ester, tryptophol, indole-3-propionate, or indole-3-butyrate. No other chemical change occurred when these substrates were incubated with the enzyme. The Km value of indolyl-3-alkane alpha-hydroxylase for L-tryptophan is 2.4 X 10(-6) M, at pH 7.2. The enzyme is inhibited by potassium cyanide (0.1 mM) or hydroxylamine (1mM), but not by NaBH4 (25 mM), aminooxyacetic acid (7mM), quinacrine (1 mM), chlortetracycline (1 mM), p-mercuribenzoate (0.1 mM), or ethylenediaminetetraacetate (1 mM). The plasma half-life (t1/2) of indolyl-3-alkane alpha-hydroxylase in tumor-bearing mice is approximately 25 h.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002849:Chromatography, Gas; D003460:Crystallization; D006418:Heme / Q000032:analysis; D006420:Hemeproteins / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007211:Indoles; D007501:Iron / Q000032:analysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000378:metabolism
D000596:Amino Acids; D006420:Hemeproteins; D007211:Indoles; D006418:Heme; D014364:Tryptophan; D007501:Iron; D006899:Mixed Function Oxygenases