Detection and identification of transient intermediates in the reactions of tryptophan synthase with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Evidence for a tetrahedral (gem-diamine) intermediate.
The reactions of 2,3-dihydro-L-tryptophan (DHT) and oxindolyl-L-alanine (OXA) with tryptophan synthase have been investigated by rapid-scanning stopped-flow (RSSF) spectroscopy and by the concentration dependence of rates measured by single-wavelength stopped-flow (SWSF) spectroscopy. The RSSF spectral changes for DHT and OXA show the disappearance of the internal aldimine (lambda max 412 nm), the formation and decay of intermediates absorbing less than or equal to 340 nm, and the appearance of the quinonoid (lambda max 492 and 480 nm, respectively). Rate constants determined by SWSF were either well resolved (i.e., k1[DHT], k-1 greater than k2, k-2 greater than k3, k-3) or indicative of a tightly coupled system (i.e., k1[OXA], k-1 greater than or equal to k2, k-2 greater than k3, k-3). The RSSF spectral changes and SWSF kinetic studies together with computer simulations of the kinetic time courses are consistent with a mechanism that includes formation of a bleached species. Detection of these shorter wavelength species in the reactions of OXA and DHT indicates that substrate analogues with tetrahedral geometry at C-3 induce new protein-substrate interactions that result in the accumulation of species not previously detected in the tryptophan synthase system. The bleached species with lambda max less than or equal to 340 nm are proposed as the gem-diamine intermediates.
Roy
M
M
Department of Biochemistry, University of California, Riverside 92521.
Miles
E W
EW
Phillips
R S
RS
Dunn
M F
MF
D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism; D003959:Diamines; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D000078183:Oxindoles; D011485:Protein Binding; D013053:Spectrophotometry / Q000379:methods; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000378:metabolism; D014367:Tryptophan Synthase / Q000378:metabolism*
D003959:Diamines; D000078183:Oxindoles; C044832:oxindolylalanine; C044338:2,3-dihydrotryptophan; D014364:Tryptophan; D014367:Tryptophan Synthase; D000409:Alanine