The Binding of Indole to the α‐Subunit and β₂‐Subunit and to the α₂β₂‐Complex of Tryptophan Synthase from Escherichia coli

Abstract

The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual a and P,-subunits and to the a2 8,-cornplex of tryptophan synthase was studied by equilibrium dialysis. The use of ['4C]indole and indolepropanol [32P]phosphate permitted simultaneous binding studies to be carried out. Competition between indole and indolepropanol phosphate in binding to a particular site was taken as evidence for that site being part of the active site of the a-subunit.The binding of indole to the active site of the a-subunit is weak (Kd = 18 mM). A second distinct site binds indole more strongly (Kd = 1.5 mM) and interacts with the active site indirectly. It is therefore designated an effector site. Furthermore, the binding of indole and/or indolepropanol phosphate appears to stabilize different conformations of the a-subunit. The B,-subunit binds indole only weakly (Kd = 12 mM) to many (n = 10) sites per polypeptide chain. The a,P,-cornplex retains one or two sites per ap-equivalent of relatively high affinity (Kd = 1.2 mM). The active sites of the component a and 8-subunits probably belong to the second class of many (n = 40) sites of low (Kd = 30 mM) affinity for indole. These findings support conclusions from the literature that both bi-substrate reactions involving indole catalyzed by tryptophan synthase and its subunits must follow strictly ordered addition mechanisms with the respective other substrate adding first. +L-tryptophan + H20. (reaction 3 )The a-subunit is capable of catalyzing reaction (2) and the P,-subunit reaction (3) albeit only 1 -2 % as efficiently as the a,P,-complex. Reactions (2) (3) can formally be considered to be partial reactions of the physiologically relevant reaction (I), which can be catalyzed only by the native a,P,-complex [I]. This hypothesis implies that indole is a free intermediate. However, no indole could be detected in various trapping experiments designed to prove its transient accumulation in the course of the reaction (1) [ 2 , 3 ] . These findings have been interpreted in terms of a channelling effect, whereby the particular organization of a hypothetical composite active site [4] precludes the equilibration of indole with the bulk solvent. However, this is not a unique explanation and more direct evidence pertaining to the relationship between the partial reactions (2) and ( 3 ) and the overall reaction (1) appears desirable. In particular, it is pertinent to ask whether the indole subsites in the active sites of the a and the P2-subunits are retained and/or modified in the a,~,-complex. Such studies would also provide ancillary evidence for interpreting steady-state kinetic data.

Steady‐State Kinetic Studies of the Synthesis of Indoleglycerol Phosphate Catalyzed by the α Subunit of Tryptophan Synthase from Escherichia coli

“…Moreover spectrophotometric titrations employing circular dichroism have shown that D-glyceraldehyde 3-phosphate binds to the free a subunit [7]. In contrast the enzyme has only weak affinity for indole at the active center [6]. In this respect the data conform to the predictions of a sequential ordered addition (nonequilibrium) mechanism with D-glyceraldehyde 3-phosphate binding first.…”

“…This illustrates the role of the substrate bound first in generating a site of relatively high affinity for indole. The numerical value of the effector indole dissociation constant K agrees with the estimate from direct binding studies and was actually used for the quantitative analysis as a more dependable value [6]. It is interesting to note that although a binding site for indole with relative high affinity exists also in native tryptophan synthase, no co-operative effects of indole are observed.…”

“…These effects seem to be exerted via conformational changes and may thus be important for catalysis [4]. There are two distinct indole-binding sites on the a subunit, only one of which overlaps with the binding site of indolepropanol phosphate [6]. This fact provides the basis for interpreting the co-operative effects exerted by indole on the initial velocity of synthesis of indoleglycerol phosphate.…”

“…However, the existence of an effector site for indole (alternative e) has been demonstraked by direct binding studies. It is distinct from (but interacts with) the indole subsite in the active center of the M subunit [6]. The simplest explanation for the observed co-operative effects is that indole bound to the effector site leads to a transition of the a subunit from a state of low to one of slightly higher activity, designated a and b respectively.…”

“…Apparent co-operativity observed with monomeric enzymes possessing a single active site can be accounted for either by (a) protein association linked to substrate binding [8-lo], (b) mechanisms involving at least three different conformational states of the enzyme under special conditions ('mnemonical transition' [1 l]), (c) branched pathways (e.g., sequential ordered addition and substitution, or ping-pong, mechanisms in parallel [12,13]), (d) the existence of two different states of the enzyme equilibrating slowly ('hysteresis' [8,9,141) or by (e) the existence of a second binding site for the substrate which interacts with the active center [15]. Possibility (a) is ruled out because neither indole [6] nor indolepropanol phosphate [5] promote association of the monomeric a subunit. Although the catalytic mechanism appears to follow a strict order of substrate addition (and therefore qualifies as a candidate for mnemonical transitions [ll]) the co-operativity is not observed with the first substrate (D-glyceraldehyde 3-phosphate) but with indole (cf: Fig.…”