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 catalyz es the alpha,beta-dehydrogenation of L-tryptophan derivatives. It was extracted from Chromobacterium violaceum and purified 108-fold to appa rent 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 th at the enzyme is composed of two components with molecular weight of a pproximately 74,000 and 14,000. It also contains a noncovalently bound heme prosthetic group, as judged from a typical spectrum showing maxi ma at 427, 530, and 560 nm. The catalyzed reaction is completed withou t side-product formation over a broad pH range comprised between 3 and 8. The enzyme is reoxidized at the expense of molecular oxygen by pro ducing one molecule of H2O2. Kinetic parameters for modification of N- acetyl-L-tryptophanamide were determined (K-m = 19.5 mu M; k(cat) = 45 .2 s(-1)). A Hill coefficient of about 1 suggests the absence of any c ooperative effect. As inferred from both its kinetic and stability opt imal conditions, L-tryptophan 2',3'-oxidase constitutes a promising to ol for chemical modification of tryptophanyl side chain in peptides an d proteins.