Mechanism of binding of substrate analogues to tryptophan indole-lyase: studies using rapid-scanning and single-wavelength stopped-flow spectrophotometry.
We have examined the binding of oxindolyl-L-alanine, (3R)-2,3-dihydro-L-tryptophan, L-homophenylalanine, and N1-methyl-L-tryptophan to tryptophan indole-lyase (tryptophanase) from Escherichia coli by using rapid-scanning and single-wavelength stopped-flow kinetic techniques. Rate constants for the reactions were determined by fitting the concentration dependencies of relaxations to either linear (pseudo-first-order) or hyperbolic (rapid second-order followed by slow first-order) equations. The reaction with oxindolyl-L-alanine forms a quinonoid intermediate that exhibits a strong peak at 506 nm. This species is formed more rapidly than with the other analogues (84.5 s-1) and is reprotonated very slowly (0.2 s-1). Reaction with L-homophenylalanine also forms a quinonoid intermediate with a strong peak at 508 nm, but the rate constant for its formation is slower (6.9 s-1). The reaction with L-homophenylalanine exhibits a transient intermediate absorbing at about 340 nm that decays at the same rate as the quinonoid peak forms and that may be a gem-diamine. Tryptophan indole-lyase reacts with (3R)-2,3-dihydro-L-tryptophan much more slowly to form a moderately intense quinonoid peak at 510 nm, and a transient intermediate absorbing at about 350 nm is also formed. The species formed in the reaction of N1-methyl-L-tryptophan exhibits a peak at 425 nm and a very weak quinonoid absorption peak at 506 nm, which is formed at less than 4 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Phillips
R S
RS
Department of Chemistry, School of Chemical Sciences, University of Georgia, Athens 30602.
Bender
S L
SL
Brzovic
P
P
Dunn
M F
MF
D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism; D000613:Aminobutyrates / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D000078183:Oxindoles; D011485:Protein Binding; D013053:Spectrophotometry; D013379:Substrate Specificity; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism; D014368:Tryptophanase / Q000378:metabolism*
D000613:Aminobutyrates; D001426:Bacterial Proteins; D000078183:Oxindoles; C044832:oxindolylalanine; C028129:tryptophan methyl ester; C044338:2,3-dihydrotryptophan; C014328:2-amino-4-phenylbutyric acid; D014364:Tryptophan; D014368:Tryptophanase; D000409:Alanine