Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism.
Tryptophan synthase, an alpha 2 beta 2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)
Anderson
K S
KS
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Miles
E W
EW
Johnson
K A
KA
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D005453:Fluorescence; D005994:Glycerophosphates / Q000378:metabolism; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D009154:Mutation; D011487:Protein Conformation; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D012694:Serine / Q000378:metabolism*; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism; D014367:Tryptophan Synthase / Q000235:genetics / Q000378:metabolism*
D005994:Glycerophosphates; D007211:Indoles; C008117:indoleglycerol phosphate; D012694:Serine; C030374:indole; D014364:Tryptophan; D014367:Tryptophan Synthase