The tryptophanase from Proteus rettgeri, improved purification and properties of crystalline holotryptophanase.
The inducible tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) was crystallized in holoenzyme from the cell extract of Proteus rettgeri. The purification procedure included ammonium sulfate fractionation, heat treatment at 60 degrees C, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystallization was performed by the addition of ammonium sulfate to the purified enzyme solution containing 20% (v/v) glycerol, 0.1 mM pyridoxal phosphate and 10 mM mercaptoethanol. The crystallized enzyme was yellow and showed absorption maxima at 340 and 420 nm. The crystalline holotryptophanase preparation was homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight of the enzyme was calculated as approx. 222 000. The amount of pyridoxal phosphate bound to the enzyme was determined to be 4 mol per mol of the enzyme. The enzyme is composed of four subunits of identical molecular size (mol. wt 55 000) and irreversibly dissociates into these subunits in the presence of a high concentration of sodium dodecylsulfate or guanidine hydrochloride. The NH2-terminal amino acid of the enzyme was identified as alanine.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate / Q000494:pharmacology; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D004355:Drug Stability; D004589:Electrophoresis, Disc; D005990:Glycerol / Q000494:pharmacology; D006146:Guanidines; D006882:Hydroxyapatites; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D008190:Lyases / Q000302:isolation & purification*; D008970:Molecular Weight; D011485:Protein Binding; D011511:Proteus / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate; D013997:Time Factors; D014368:Tryptophanase / Q000302:isolation & purification* / Q000378:metabolism; D014461:Ultracentrifugation
D000596:Amino Acids; D006146:Guanidines; D006882:Hydroxyapatites; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D012967:Sodium Dodecyl Sulfate; D008190:Lyases; D014368:Tryptophanase; D005990:Glycerol; D000645:Ammonium Sulfate