1 Tryptophan Synthetase

Yanofsky, Charles; Crawford, Irving P.

doi:10.1016/s1874-6047(08)60445-x

Cited by 128 publications

(116 citation statements)

References 86 publications

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“…The unusually high affinity (50°/, inhibition a t 0.05 mM indole acrylic acid (cf. 0.35 mM for the Neurospora enzyme 1231) suggests a special role of this inhibitor in reaction mechanism (B), possibly interference with the aminoacrylic-enzyme complex postulated as the terminal step of tryptophan formation [1,22].…”

Section: Studies On the Mechanism Of The Enzymatic Inactivation Of Trmentioning

confidence: 99%

Tryptophan Synthase from Yeast

Wolf

Hoffmann

1974

European Journal of Biochemistry

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Tryptophan synthase was purified from Xaccharomyces cerevisiae with the aid of affinity chromatography up to a specific activity of 525 units/g protein. Electrophoresis on polyacrylamide gel showed one main protein component. Gel filtration on Sephadex G-150 indicates an average molecluar weight of 143 000. Dissociation with dodecylsulfate followed by dodecylsulfate-acrylamide gcl electrophoresis leads to the conclusion that the enzyme is probably cornposed of four subunits of equal size. The Km-values with respect to its substrates indoleglycerol phosphate, indole and L-serine were estimated. L-Serine was shown to increase the K,-value for indoleglycerol phosphate about 7-fold. Several indole analogues inhibit the tryptophan synthase reaction (B) (i.e. indole + serine --f tryptophan); indoleacrylic acid is the most effective. The pH-optimum of tryptophan synthase is around 6.9 to 7.0. Sulfhydryl modification by 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuriphenylsulfonic acid results in a large loss of tryptophan synthase activity, which cannot be prevented by the substrates L-serine and indole. The tryptophan synthase inactivating system from yeast affects the catalysis of both reactions (A) (i.e. indoleglycerol phosphate + indole + glyceraldehyde 3-phosphate) and (B).Tryptophan synthase (L-serine hydrolyase, adding indole) catalyzes the final reaction in tryptophan hiosynthesis :In all microorganisms in which i t has been studied, the enzyme is also capable of catalyzing the following two half reactions [ 11 :Indole-glycerol-P -+ Indole + n-glyceraldehyde-3-P.Pyridoxal phosphate is required as a coenzyme in reactions (AB) and (B). The Escherichia coli and Neurospora crassa enzymes appear to convert indoleglycerol phosphate to tryptophan (the sum of reactions A and B) without releasing free indole from the enzymc [2,3]. This mechanism predominates also with the Xaccharomyces cerevisiae enzyme, but significant amounts of free indole are also formed [4]. In bacteria tryptophan synthase can bc dissociated Abbreviations. 5,5'-Dithiobis(2-nitrobenzoic acid), Nbs,; p-chloromercuriphenylsulfonic acid, pCI-HgBzOH.Enzymes. Tryptophan synthase (or r,-serine hydrolyase (adding indole) (EC 4.2.1.20). __.-readily and reversibly into two distinct components designated A and B [l], the A protein catalyzing reaction (A), the B protein reaction (B). The rates of reactions catalyzed by the individual components, however, are, in general, much lower than that catalyzed by the AB complex. In contrast, the enzyme from the fungus Neurospora is not dissociable and acts only as the whole unit, though i t is composed of two pairs of polypeptide chains [5,6].Genetic studies have been carried out [4] with the fungus S. cerevisiae pertainent to the structure of tryptophan synthase and its reaction mechanism. It was shown also by Manney 17 J and by Katsunuma et al. [S] that the yeast enzyme is inactivated in an enzymatic process. The inactivating system consists of two proteolytic enzymes and associated inhibitors [9,9a,Bb]. Th...

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Section: Studies On the Mechanism Of The Enzymatic Inactivation Of Trmentioning

confidence: 99%

Tryptophan Synthase from Yeast

Wolf

Hoffmann

1974

European Journal of Biochemistry

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“…Creighton and Yanofsky (1966) have shown that the ratio of the molar specific activities in the B reaction of 01202/32 = 2 under assay conditions. Moreover, the ratio for eA22/fl2 equals 50 (Yanofsky and Crawford, 1972 complex was mixed with an excess of holo02 subunit, and enzymic activity increased exponentially with incubation time. The rate constants of release of a subunit from both a2holoj32 and a2 reduced i32 are about the same (cf .…”

Section: Introductionmentioning

confidence: 98%

“…Kinetic studies of the mechanism of assembly makes it possible to investigate the following questions: (a) can one detect first-order processes, which may be related to changes of subunit conformation, and (b) thase from Escherichia coli as a convenient system (Yanofsky and Crawford, 1972;Miles, 1979). This a2(2 complex is readily dissociated into two a subunits and one tightly associated (2 subunit.…”

Section: Introductionmentioning

confidence: 99%

The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli.

Lane¹,

Paul²,

Kirschner³

1984

The EMBO Journal

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The a subunit is bound with negative cooperativity to the holo(2 subunit of tryptophan synthase in phosphate buffer.Thus it is feasible to measure separately the rates of formation both of the stable aC32 subcomplex from O2, and of the mature a2f32 complex from rn2, using stopped-flow techniques. Addition of each a subunit proceeds in two steps; an initial cx, protomer is formed rapidly, which subsequently isomerizes slowly to the equilibrium state. The rates of dissociation of both the 0a2 and a2/2 complexes were measured by trapping released a subunit with enzymically inactive reduced (2 subunit. The reversal of the slow isomerization both determines the rate of dissociation, and accounts for the high overall affinity of the (3 protomer for the a subunit. The data fit to a sequential assembly mechanism consisting of seven protein species and yields values for most of the rate constants and all of the microscopic equilibrium constants. Negative cooperativity arises from a weaker initial binding of the second a subunit, as expressed by its larger off-constant, possibly due to steric hindrance. The kinetics of binding of L-erine and indolepropanol phosphate during the assembly process shows that the (3 protomer is already partially activated in the initial a(3 complex. Full activation is achieved in the slow isomerization reaction. In contrast, the a subunit gains high affinity for indolepropanol phosphate only in the isomerization reaction.These observations indicate that the isomerization involves synchronous conformation changes of both a and (3 protomers.

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“…The bacterial tryptophan synthase ␣ 2 ␤ 2 complex catalyzes the last two steps in the biosynthesis of L-tryptophan (1)(2)(3)(4). Indole is formed from the cleavage of indole-3-glycerol phosphate in the ␣-active site and subsequently is channeled, via a hydrophobic tunnel, to the ␤-active site (5,6) where it is combined with L-Ser to make L-Trp.…”

mentioning

confidence: 99%

“…In the crystal, this quinonoid does not accumulate appreciably (29). However, it is possible to form quinonoid species that are analogous to E(Q 3 ) by reacting the aminoacrylate intermediate with nucleophiles such as ␤-mercaptoethanol (␤-MSH) 1 (30), indoline, phenylhydrazine, aniline, and small organic amines (31,32). Here, we have mainly investigated the accumulation and the stability of the quinonoid species formed by using the indole analog indoline in solution and crystal.…”

mentioning

confidence: 99%

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

Mozzarelli¹,

Peracchi²,

Rovegno³

et al. 2000

Journal of Biological Chemistry

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Quinonoid intermediates play a key role in the catalytic mechanism of pyridoxal 5-phosphate-dependent enzymes. Whereas the structures of other pyridoxal 5-phosphate-bound intermediates have been determined, the structure of a quinonoid species has not yet been reported. Here, we investigate factors controlling the accumulation and stability of quinonoids formed at the ␤-active site of tryptophan synthase both in solution and the crystal. The quinonoids were obtained by reacting the ␣-aminoacrylate Schiff base with different nucleophiles, focusing mainly on the substrate analogs indoline and ␤-mercaptoethanol. In solution, both monovalent cations (Cs ؉ or Na ؉ ) and alkaline pH increase the apparent affinity of indoline and favor accumulation of the indoline quinonoid. A similar pH dependence is observed when ␤-mercaptoethanol is used. As indoline and ␤-mercaptoethanol exhibit very distinct ionization properties, this finding suggests that nucleophile binding and quinonoid stability are controlled by some ionizable protein residue(s). In the crystal, alkaline pH favors formation of the indoline quinonoid as in solution, but the effect of cations is markedly different. In the absence of monovalent metal ions the quinonoid species accumulates substantially, whereas in the presence of sodium ions the accumulation is modest, unless ␣-subunit ligands are also present. ␣-Subunit ligands not only favor the formation of the intermediate, but also reduce significantly its decay rate. These findings define experimental conditions suitable for the stabilization of the quinonoid species in the crystal, a critical prerequisite for the determination of the three-dimensional structure of this intermediate.The bacterial tryptophan synthase ␣ 2 ␤ 2 complex catalyzes the last two steps in the biosynthesis of L-tryptophan (1-4). Indole is formed from the cleavage of indole-3-glycerol phosphate in the ␣-active site and subsequently is channeled, via a hydrophobic tunnel, to the ␤-active site (5, 6) where it is combined with L-Ser to make L-Trp. The reaction at the ␤-active site depends on the cofactor pyridoxal 5Ј-phosphate and proceeds through the formation of several intermediates, which are characterized by distinct absorption properties (Scheme 1) (2, 7-10).The ␣-and ␤-subunit activities are allosterically regulated (2-4, 6, 11-20) via the selective stabilization of ␣-and ␤-subunit conformations consisting of an "open," catalytically inactive state, and a "closed" catalytically active state (11,12,21,22). 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, 12-14). 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 cl...

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1 Tryptophan Synthetase

Cited by 128 publications

References 86 publications

Tryptophan Synthase from Yeast

Tryptophan Synthase from Yeast

The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli.

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

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