Purification and partial characterization of an amino acid alpha,beta- dehydrogenase, L-tryptophan 2',3'-oxidase from Chromobacterium violaceum.
L-Tryptophan 2',3'-oxidase is a novel enzyme that specifically catalyzes the alpha,beta-dehydrogenation of L-tryptophan derivatives. It was extracted from Chromobacterium violaceum and purified 108-fold to apparent homogeneity with a 34% overall recovery. The molecular weight of the native enzyme is approximately 680,000, and its isoelectric point is nearly equal to 4. SDS-polyacrylamide gel electrophoresis showed that the enzyme is composed of two components with molecular weight of approximately 74,000 and 14,000. It also contains a noncovalently bound heme prosthetic group, as judged from a typical spectrum showing maxima at 427, 530, and 560 nm. The catalyzed reaction is completed without side-product formation over a broad pH range comprised between 3 and 8. The enzyme is reoxidized at the expense of molecular oxygen by producing one molecule of H2O2. Kinetic parameters for modification of N-acetyl-L-tryptophanamide were determined (Km = 19.5 microM; kcat = 45.2 s-1). A Hill coefficient of about 1 suggests the absence of any cooperative effect. As inferred from both its kinetic and stability optimal conditions, L-tryptophan 2',3'-oxidase constitutes a promising tool for chemical modification of tryptophanyl side chain in peptides and proteins.
Genet
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Commissariat à l'Energie Atomique, Département d'Ingénierie et d'Etudes des Protéines, Gif-sur-Yvette, France.
Denoyelle
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Ménez
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D002384:Catalysis; D002861:Chromobacterium / Q000201:enzymology*; D004790:Enzyme Induction; D004795:Enzyme Stability; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009994:Osmolar Concentration; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism
D006420:Hemeproteins; C016952:N-acetyltryptophanamide; D014364:Tryptophan; D010088:Oxidoreductases; C088650:L-tryptophan 2',3'-oxidase