Conformational changes in the active site of tryptophanase revealed by the circular dichroism method.
Tryptophanase from E. coli displays positive CD in the coenzyme absorption bands at 337 and 420 nm. Breaking of the internal coenzyme-lysine imine bond upon reaction with hydroxylamine or amino-oxyacetate is accompanied by a strong diminution of the positive CD. Interaction of tryptophanase with L-threonine and beta-phenyl-DL-serine(threo form) leads to a decrease in absorbance at 337 nm and to an increase at 425 nm. This is associated with inversion of the CD sign, i.e. with disappearance of the positive CD in the 420-nm band and its replacement by a negative CD. L-Phenylalanine, alpha-methyl-DL-serine and D-alanine cause an increase in absorbance at 425-430 nm and a diminution of the positive CD in this band. In the presence of D-alanine and indole a negative CD appears in the 400-450 nm region. It is inferred that an external coenzyme-quasisubstrate aldimine is formed on interaction of the above amino acids with the enzyme. L-Alanine and oxindolyl-L-alanine evoke an intense narrow absorption band at 500 nm ascribed to a quinonoid intermediate; a positive CD is observed in this band. The dissymmetry factor delta A/A in the 500-nm band is much smaller than that in the absorption bands of the unliganded enzyme. Inversion of the CD sign on formation of the external aldimine and diminution of the dissymmetry factor in the quinonoid band indicate that reorientations of the coenzyme occur in the course of the catalytic action of tryptophanase.
Zakomirdina
L N
LN
Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow.
Sakharova
I S
IS
Torchinsky
Y M
YM
D000596:Amino Acids / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D003067:Coenzymes / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008190:Lyases / Q000378:metabolism*; D011487:Protein Conformation; D013053:Spectrophotometry; D014368:Tryptophanase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000596:Amino Acids; D003067:Coenzymes; D006898:Hydroxylamines; D019811:Hydroxylamine; D008190:Lyases; D014368:Tryptophanase