[Study of coenzyme reorientation in the active center of tryptophanase by linear dichroism method].
Tryptophanase from E.coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra were 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 sign and form of this band are changed upon the enzyme reaction with substrate analogs. The 490 nm band is present in the LD spectra of holo- and apoenzyme and disappears after treatment with NaBH4. It is suggested that the 490 nm band belongs to a quinoid enzyme subform. The reaction of tryptophanase with threo-beta-phenyl-DL-serine and L-threonine leads to formation of the external aldimine with a strong absorption band at 420-425 nm. The reduced LD (delta A/A) in this band is one order of magnitude greater than that in the 420 nm of the free enzyme. The complex with D-alanine is characterized by an intermediate LD value in the 425 nm band. In the presence of indole this complex displays the same LD as that observed with beta-phenylserine. The reaction of tryptophanase with L-alanine and oxindolyl-L-alanine leads to formation of the quinoid intermediate with a 500 nm absorption band. The LD value in this band differs from those in the absorption bands of the free enzyme. It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction.
Zakomyrdina
L N
LN
Sakharova
I S
IS
Torchinskiĭ
Iu M
IuM
D001665:Binding Sites; D003067:Coenzymes / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D008190:Lyases / Q000032:analysis*; D013057:Spectrum Analysis / Q000379:methods; D013379:Substrate Specificity; D014368:Tryptophanase / Q000032:analysis*
D003067:Coenzymes; D008190:Lyases; D014368:Tryptophanase
Issledovanie reorientatsiĭ kofermenta v aktivnom tsentre triptofanazy metodom lineĭnogo dikhroizma.