Linear dichroism studies of tryptophanase and its quasisubstrate complexes oriented in polyacrylamide gel.
Tryptophanase from Escherichia coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra have been measured. The free enzyme displays four LD bands at 305, 340, 425 and 490 nm. Two bands at 340 and 425 nm belong to the internal coenzyme-lysine aldimine. The 305-nm band apparently belongs to an aromatic amino acid residue. The 490-nm band disappears after treatment with NaBH4 or after incubation with L-alanine and subsequent dialysis. It is suggested that the 490-nm band belongs to a quinonoid enzyme subform. The reaction of tryptophanase with threo-3-phenyl-DL-serine, L-threonine and D-alanine leads to formation of an external aldimine with an intense absorption band at 420-425 nm. The values of reduced LD (delta A/A) in this band strongly differ from that in the 420-nm band of the free enzyme. The LD value of the complex with D-alanine is intermediate between those of the free enzyme and the complex with 3-phenylserine. In the presence of indole the complex with D-alanine displays the same LD as that observed with 3-phenylserine. The reaction of tryptophanase with L-alanine or oxindolyl-L-alanine leads to formation of a quinonoid intermediate with an absorption band near 500 nm. The LD value in this band is close to that of an external aldimine with L-threonine. It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction.
Zakomirdina
L N
LN
V. A. Engelhardt Institute of Molecular Biology, Academy of Sciences, Moscow, USSR.
Sakharova
I S
IS
Torchinsky
Y M
YM
D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D008024:Ligands; D000078183:Oxindoles; D011485:Protein Binding; D011487:Protein Conformation; D012694:Serine / Q000031:analogs & derivatives; D013057:Spectrum Analysis; D013912:Threonine; D014368:Tryptophanase / Q000737:chemistry / Q000648:ultrastructure*
D008024:Ligands; D000078183:Oxindoles; D013912:Threonine; C044832:oxindolylalanine; D012694:Serine; D014368:Tryptophanase; D000409:Alanine