Subunit structure of the B component of Escherichia coli tryptophan synthetase

Hathaway, Gary M.; Kida, Setsuko; Crawford, Irving P.

doi:10.1021/bi00831a032

Cited by 54 publications

(28 citation statements)

References 21 publications

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“…Thus, the confirmation of the "globule"-model proposed to account for the folding of E. coli f3-D-galactosidase (33) required the very tedious and effort-consuming (but how elegant) immunological approach of Celada et al (34). For nicked 132, both difficulties are likely to be easily overcome, since the renaturation of intact 132 has been described as Biochemistry: Hogberg-Raibaud and Goldberg straightforward and easy (4) and since the protein carries manv specific interaction sites that can serve as probes for the conformatiot of the "renatured" isolated fragments.…”

Section: Resultsmentioning

confidence: 99%

Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

Högberg-Raibaud¹,

Goldberg²

1977

Proc. Natl. Acad. Sci. U.S.A.

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Globular proteins often appear to consist of distinct compact "domains," and the assumption is frequently implicitly made that these domains correspond to intermediate structures in the folding process. If this assumption is correct, the polypeptide fragment that builds up a domain should be able to spontaneously fold into its native conformation even when isolated. In an attempt to isolate and study such a fragment, the #2 subunit of tryptophan synthetase [

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Section: Resultsmentioning

confidence: 99%

Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

Högberg-Raibaud¹,

Goldberg²

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…In order to overcome the problem of proteolytic degradation during the timeconsuming fractionation procedures, we developed a new strategy for purification which avoids the continuous interaction of proteases with the enzyme by fractionation under denaturing conditions and subsequent reconstitution. Based on earlier reactivation experiments with oligomeric enzymes [34-361 it has been shown recently that E. coli tryptophan synthase f l~ subunit can be reversibly denatured in the presence of 2 4 M urea [37] or 4.5 M guanidine . HCl at acidic pH [38].…”

Section: Discussionmentioning

confidence: 99%

Purification of Tryptophan Synthase from Saccharomyces cerevisiae and Partial Activity of Its Nicked Subunits

1979

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Enzymes can be reconstituted with high yield after dissociation and deactivation in strong denaturants. This finding can be utilized to separate proteases and other impurities (with molecular weights differing under denaturing conditions from the subunit molecular weight of the enzyme to be purified) from the desired enzyme. Applying this approach to tryptophan synthase from yeast, a homogeneous preparation with a molecular weight of 154000 Jr 5000 and a specific activity of 1200 & 100 Yanofsky units/mg has been obtained. The latter value exceeds the previously reported maximum activity by a factor of 10, and characterizes the enzyme in its native state. The K , values for D-glyceraldehyde 3-phosphate and indole of the indole-glycerolphosphate synthesis (reaction 2 ) and the pH optimum, as well as the K,,, values for L-serine, indole, and pyridoxal5'-phosphate of the L-tryptophan condensation (reaction 3) have been determined.At least two tryptophan-synthase-inactivating proteinases from yeast have been well characterized in the past. Therefore, the low activity of previous preparations may be assumed to be caused by proteolytic cleavage. To prove this hypothesis, tryptophan synthase was prepared following a conventional procedure. Using ultracentrifugation and gel chromatography under quasi-physiological conditions, this enzyme has been found to be a homogeneous species of M , = 75000 f 3000 and = 3.3 k 0.2 S, which is still capable of catalyzing both reactions 2 and 3 with a specific activity for the latter reaction of 120 Yanofsky units/mg. Comparing the K, values for all relevant ligands, only the K, value for pyridoxal 5'-phosphate shows a marked difference with respect to the value observed for the native enzyme. At -18 "C the nicked enzyme is stable over a period of several weeks.As measured by sedimentation equilibrium in 6M guanidine . HCl at pH 2.3, the molecule consists of two large polypeptide fragments, M,,I = 22000 k 3000 and Mr,2 = 50000 + 5000.These results suggest tryptophan synthase from yeast to be a dimer of two bifunctional subunits; obviously the specific proteinases preferentially attack a region forming a link between two domains of the enzyme corresponding to the ci and / 3 subunits of the Escherichia coli enzyme.Tryptophan synthase isolated from different microorganisms catalyzes the last step in tryptophan biosynthesis :

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“…Reduced 02 subunit was prepared according to Hathaway et al (1969), using 5 M NaBH4 in 0.05 N NaOH (Raibaud and Goldberg, 1973) rather than solid NaBH4 as the reducing agent.…”

Section: Materials and Methods Materialsmentioning

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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Subunit structure of the B component of Escherichia coli tryptophan synthetase

Cited by 54 publications

References 21 publications

Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

Preparation and characterization of a modified form of beta2 subunit of Escherichia coli tryptophan synthetase suitable for investigating protein folding.

Purification of Tryptophan Synthase from Saccharomyces cerevisiae and Partial Activity of Its Nicked Subunits

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

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