Allosteric linkages between .beta.-site covalent transformations and .alpha.-site activation and deactivation in the tryptophan synthase bienzyme complex

Abstract

This work examines two aspects of the catalytic mechanism and allosteric regulation of the tryptophan synthase bienzyme complex from Salmonella typhimurium: (a) the chemical mechanism by which indole and other nucleophiles react with the enzyme-bound alpha-aminoacrylate Schiff base intermediate, E(A-A), to form quinonoidal intermediates, E(Q), and (b) the effects of covalent transformations at the beta-site on the catalytic activity of the alpha-site. Transient kinetic studies in combination with alpha-secondary deuterium isotope effects are undertaken to determine the mechanism of nucleophile addition to E(A-A). These studies establish that nucleophilic attack is best described by a two-step reaction sequence consisting of a binding step that is followed by Michael addition to the conjugated double bond of E(A-A). Analysis of isotope effects suggests that the transition state for indole addition gives an E(A-A) beta-carbon that resembles an sp3 center, while the stronger nucleophiles, indoline and beta-mercaptoethanol, have transition states that appear to more closely resemble an sp2 beta-carbon. The effects of beta-site covalent transformations on alpha-site catalysis were studied using quasi-stable beta-site intermediates and the alpha-site substrate analogue 3-[6-nitroindole]-D-glycerol 3'-phosphate (6-nitro-IGP). It was found that the cleavage of 6-nitro-IGP is strongly activated by the formation of E(A-A) and various E(Q) species at the beta-site but not by external aldimine species. Therefore, we conclude that the conversion of the L-Ser external aldimine to E(A-A) is the beta-site process which activates the alpha-site, while conversion of E(Q) to the L-Trp external aldimine triggers deactivation of the alpha-site. These findings are discussed within the context of allosteric regulation of substrate channeling in tryptophan synthase catalysis.

Effect of pH and Monovalent Cations on the Formation of Quinonoid Intermediates of the Tryptophan Synthase α2β2 Complex in Solution and in the Crystal

“…13) and for ␤-MSH (pK a ϳ9.5; Ref. 41), it is unlikely that quinonoid formation is controlled by the ionization properties of the nucleophile.…”

“…Different protein conformations are associated with E (Aex 1 ) and E(A-A), the former residing in a partially open state and the latter in the closed state. Both E(Q H ) Indoline / E(Q) Indoline and E(A-A) are predominantly in a closed conformation (13,14,22).…”

“…Only the closed state of the ␤-subunit appears to be competent in the transmission of allosteric signals and, therefore, in stabilizing the closed, catalytically active conformation of the ␣-subunit. Through this mechanism, the catalytic activities of ␣-and ␤-subunits are finely tuned and kept in phase (4,12,13,22).Determining the three-dimensional structure of an enzyme at different stages of the catalytic process is an important component of the effort to understand the structural basis of catalysis. In the case of tryptophan synthase, this is even more important since it would help to define the interplay between catalysis and regulation.…”

“…Only the closed state of the ␤-subunit appears to be competent in the transmission of allosteric signals and, therefore, in stabilizing the closed, catalytically active conformation of the ␣-subunit. Through this mechanism, the catalytic activities of ␣-and ␤-subunits are finely tuned and kept in phase (4,12,13,22).…”

“…The internal and the external aldimines in the ␤-active site are in open and partially open conformations, respectively, and do not send regulatory signals to the ␣-active site (4,(12)(13)(14). The ␣-aminoacrylate, E(A-A), exists both in an open and a closed conformation depending on the presence of monovalent cations, whereas the quinonoid, E(Q 3 ), is predominantly in the closed state (13). Only the closed state of the ␤-subunit appears to be competent in the transmission of allosteric signals and, therefore, in stabilizing the closed, catalytically active conformation of the ␣-subunit.…”