Interactions of Escherichia coli tryptophanase with quasisubstrates and monovalent cations studied by the circular dichroism and fluorescence methods.
The reaction of tryptophanase and its W330F and W248F mutant forms with quasi-substrates forming an external pyridoxal phosphate aldimine or quinonoid is accompanied by the appearance of a positive circular dichroism (CD) peak at 290 nm. The peak seems to arise from a Tyr residue undergoing reorientation during the reaction. The peak does not appear upon formation of non-covalent Michaelis complexes of the enzyme with quasi-substrates such as indolepropionate, beta-phenyllactate and alpha-methylphenylalanine. The non-covalent complexes and external aldimines exhibit similar absorption spectra but can be distinguished by their CD and by the intensity of their fluorescence. Formation of the non-covalent complexes leads to an increase in positive CD at 420 nm while formation of the external aldimines leads to disappearance of the positive CD at 420 nm and its replacement by negative CD; it also leads to strong quenching of the coenzyme fluorescence at 500 nm. The quantum yield of fluorescence of the external aldimines is 6-times lower than that of the internal aldimine. Activating cations (K+, NH4+) strongly diminish the intensity of a negative protein CD band at 275 nm. From a comparison of the intensity of this band in the spectra of the wild-type holo- and apoenzyme and in the tryptophan mutants, it was deduced that the band belongs to a Tyr residue, which may be a part of the cation-binding site or located in its immediate vicinity.
Ben-Kasus
T
T
Department of Chemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Markel
A
A
Gdalevsky GYa
Torchinsky
Y M
YM
Phillips
R S
RS
Parola
A H
AH
D001665:Binding Sites; D002414:Cations, Monovalent / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism*; D014368:Tryptophanase / Q000737:chemistry* / Q000378:metabolism*
D002414:Cations, Monovalent; D011994:Recombinant Proteins; D014364:Tryptophan; D014368:Tryptophanase