Monovalent Cations Partially Repair a Conformational Defect in a Mutant Tryptophan Synthase α2β2 Complex (β-E109A)

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The mechanism of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium is explored by determining the effects of pH, of temperature, and of isotopic substitution on the pyridoxal phosphate-dependent reaction of L-serine with indole to form L-tryptophan. The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in substrate binding and catalysis with pK a 1 ‫؍‬ 6.5, pK a 2 ‫؍‬ 7.3, and pK a 3 ‫؍‬ 8.2-9. A significant primary isotope effect (ϳ3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that the base that accepts the ␣-proton (␤Lys-87) is protonated at low pH, slowing the abstraction of the ␣-proton and making this step at least partially rate-limiting. pK a 2 is assigned to ␤Lys-87 on the basis of the kinetic isotope effect results and of the observation that the competitive inhibitors glycine and oxindolyl-L-alanine display single pK i values of 7.3. The residue with this pK a (␤Lys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. Investigations of the temperature dependence of the pK a values support the assignment of pK a 2 to ␤Lys-87 and suggest that the ionizing residue with pK a 1 could be a carboxylate, possibly ␤Asp-305, and that the residue associated with a conformational change at pK a 3 may be ␤Lys-167. The occurrence of a closed to open conformational conversion at high pH is supported by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates.The tryptophan synthase ␣ 2 ␤ 2 complex (EC 4.1.2.20) is a useful system for investigating relationships between protein structure and function (for reviews, see Refs. 1-4). The separate tryptophan synthase ␤ 2 subunit 1 and the ␤ subunit in the ␣ 2 ␤ 2 complex catalyze the pyridoxal phosphate (PLP) 2 -dependent conversion of L-serine and indole to L-tryptophan. A number of intermediates in this reaction have been identified from spectroscopic and kinetic studies (see Scheme I in Discussion). The three-dimensional structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium (5) revealed that the active sites of the ␣ and ␤ subunits are ϳ25 Å apart and are connected by a hydrophobic tunnel. This tunnel serves as a passageway for indole from the active site of the ␣ subunit, where it is produced by cleavage of indole-3-glycerol phosphate, to the active site of the ␤ subunit, where it reacts with L-serine to form L-tryptophan. The activity at each site is modulated by reciprocal communication between the ␣ and ␤ sites. These heterotrophic interactions are proposed to switch the ␣ and ␤ subunits between "open" (catalytically inactive) and "closed" (catalytically active) conformations, to coordinate the activities at the two sites, and to prevent the escape of indole (4). Recent crystallographic results provide direct evidence for ligand-induced conformational changes that result in physical closure of loop structures in the ␣ subunit a...

Section: Discussionmentioning

confidence: 89%

Catalytic Mechanism of the Tryptophan Synthase α2β2 Complex

Ro¹,

Miles

1999

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“…2A, right panel, peaks B and C, Fig. 3A) to ␤Glu 109 , which is believed to play a crucial role in the catalytic activity and the substrate specificity of the TRPS ␤-reaction (42,43). This may explain the 2-fold lower ␤ activity of the ␤S178P mutant compared with the wild-type enzyme (21).…”

Section: Ligand-free ␤S178p Structurementioning

confidence: 95%

Crystal Structure of the βSer178 → Pro Mutant of Tryptophan Synthase

Weyand¹,

Schlichting²,

Herde³

et al. 2002

The catalytic activity of the pyridoxal 5-phosphatedependent tryptophan synthase ␣ 2 ␤ 2 complex is allosterically regulated. The hydrogen bond between the helix ␤H6 residue ␤Ser 178 and the loop ␣L6 residue Gly 181 was shown to be critical in ligand-induced intersubunit signaling, with the ␣-␤ communication being completely lost in the mutant ␤Ser 178 3 Pro (Marabotti, A., De Biase, D., Tramonti, A., Bettati, S., and Mozzarelli, A. (2001) J. Biol. Chem. 276, 17747-17753). The structural basis of the impaired allosteric regulation was investigated by determining the crystal structures of the mutant ␤Ser 178 3 Pro in the absence and presence of the ␣-subunit ligands indole-3-acetylglycine and glycerol 3-phosphate. The mutation causes local and distant conformational changes especially in the ␤-subunit. The ligand-free structure exhibits larger differences at the N-terminal part of helix ␤H6, whereas the enzyme ligand complexes show differences at the C-terminal side. In contrast to the wild-type enzyme loop ␣L6 remains in an open conformation even in the presence of ␣-ligands. This effects the equilibrium between active and inactive conformations of the ␣-active site, altering k cat and K m , and forms the structural basis for the missing allosteric communication between the ␣-and ␤-subunits.The ␣ 2 ␤ 2 complex of tryptophan synthase (TRPS) 1 (EC 4.2.1.20) is a bifunctional enzyme that is considered a paradigm for the analysis of intersubunit regulatory signals. The enzyme is formed by two ␣-and ␤-subunits, arranged in a linear ␣␤␤␣ geometry (1), and catalyzes the last two steps in the biosynthesis of L-tryptophan in a highly concerted mode. The isolated ␣-and ␤-subunits exhibit an activity about 2 orders of magnitude lower that in the tetramer, indicating that the formation of the complex is accompanied by subunit conformational changes (2, 3). Further levels of regulation are achieved by ligand-induced intersubunit signals that are pH-, temperature-, and cation-dependent and keep the catalytic activities of ␣ and ␤-active sites in phase (4 -11). The mechanism of the ␣-␤ activation and allosteric regulation is based on an open-close transition of both subunits (7, 12, 13) involving the movement of several parts of the ␣-and ␤-subunits. In particular, signal transduction from the ␣-to the ␤-active site involves residues belonging to loop ␣L2, loop ␣L6, and the helix ␤H6 (14, 15), the latter structural element being part of the ␤Ϫsubunit COMM domain, as defined by Schneider et al. (15). The question whether there is a unique or multiple pathways of communication and whether these pathways are specialized in controlling defined functional properties of the enzyme was addressed by investigating the activity and regulatory properties of several mutants of the ␣-and ␤-subunits. It was found that mutants in loop ␣L2 and mutants in helix ␤H6 that interact with loop ␣L2 exhibit altered ␣ and ␤ activities (16 -18), whereas mutants in loop ␣L6 and mutants in helix ␤H6 interacting with loop ␣L6 also exhibit altered alloster...

“…2 Irreversible inactivation occurs at the higher temperature (T i ϭ ϳ77°C) in the presence of a higher salt concentration that causes protein aggregation (18,19). Other studies show that the low temperature transition results in a small, low temperature endotherm in differential scanning calorimetry, perturbation in the environment of Trp-177 but not of tyrosine residues, and loss in the ellipticity of PLP (15-17).…”

mentioning

confidence: 99%

“…The results provide evidence for a reversible thermal transition in the ␤ 2 subunit at a much lower temperature (T m ϭ ϳ41-47°C) than the temperature (ϳ80°C) at which the ␤ 2 subunit undergoes a major unfolding transition (15)(16)(17). 2 Irreversible inactivation occurs at the higher temperature (T i ϭ ϳ77°C) in the presence of a higher salt concentration that causes protein aggregation (18,19). Other studies show that the low temperature transition results in a small, low temperature endotherm in differential scanning calorimetry, perturbation in the environment of Trp-177 but not of tyrosine residues, and loss in the ellipticity of PLP (15)(16)(17).…”

mentioning

confidence: 99%

A Thermally Induced Reversible Conformational Transition of the Tryptophan Synthase Subunit Probed by the Spectroscopic Properties of Pyridoxal Phosphate and by Enzymatic Activity

Ahmed

McPhie

Miles

1996

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A reversible thermally induced conformational transition of the ␤ 2 subunit of tryptophan synthase from Salmonella typhimurium has been detected by use of the pyridoxal 5-phosphate coenzyme as a spectroscopic probe. Increasing the temperature converts the major form of pyridoxal 5-phosphate bound to the ␤ 2 subunit from a ketoenamine species with max at 410 nm to a enolimine species with max at 336 nm (T m ‫؍‬ ϳ43°C) and results in loss of the circular dichroism signal at 410 nm and of fluorescence emission at 510 nm. The results indicate that increasing the temperature favors a conformer of the enzyme that binds pyridoxal 5-phosphate in a more nonpolar environment and leads to loss of asymmetric pyridoxal 5-phosphate binding. The internal aldimine between pyridoxal 5-phosphate and the ⑀-amino group of lysine 87 is not disrupted by increased temperature because sodium borohydride treatment of the enzyme at either 15 or 60°C results in covalent attachment of [4- Pyridoxal 5Ј-phosphate (PLP) 1 serves as the coenzyme for many enzymes that catalyze a wide variety of reactions involved in the metabolism of amino acids (racemization, transamination, ␤-elimination, ␤-replacement, decarboxylation, etc.) (1, 2). In every PLP-dependent enzyme, the PLP coenzyme forms an internal aldimine with the ⑀-amino group of a lysine residue (see structures in Fig. 1A under "Results"). This internal aldimine usually exhibits an absorption maximum at 410 -430 nm attributed to the resonance stabilized ketoenamine form II in Fig. 1A (3-5). The dipolar structure II is favored by a more polar environment. Most PLP enzymes also have peaks at 330 -340 nm, which may represent the enolimine tautomer (I in Fig. 1A), a neutral species that prefers a nonpolar environment (3-6). Reactions with substrates or inhibitors or changes in pH often lead to marked alterations in the spectrum of the enzyme-bound PLP (2, 5). Thus PLP serves as a useful chromophoric reporter group for changes in the PLP binding site in enzymes and for reactions with substrates and inhibitors.The ␤ 2 subunit of tryptophan synthase (EC 4.2.1.20) catalyzes a number of PLP-dependent reactions including a ␤-elimination reaction with L-serine (Equation 1) and a ␤-replacement reaction with L-serine and indole (Equation 2).(For reviews see Refs. 7-9.) PLP serves as a useful spectrophotometric indicator of alterations in the PLP binding site of the ␤ 2 subunit. For example, the UV-visible spectra (10, 11) and circular dichroism spectra (12, 13) of bound PLP are altered by interaction of the ␤ 2 subunit with the ␣ subunit of tryptophan synthase to form an ␣ 2 ␤ 2 complex and by certain ␤ 2 subunit mutations in the ␣ 2 ␤ 2 complex (11). The three-dimensional structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium revealed that the chains are arranged in a nearly linear ␣␤␤␣ order (14). The larger ␤ chain (43,000 M r ) in the complex has two domains of about equal size, designated the N-and C-domains, which are folded in similar helix/sheet/helix structures. The ...