Mechanism of reconstitution of the apo.beta.2 subunit and the .alpha.2apo.beta.2 complex of tryptophan synthase with pyridoxal 5'-phosphate: kinetic studies

Bartholmes, Peter; Balk, Hubert; Kirschner, Kasper

doi:10.1021/bi00560a022

Cited by 27 publications

(26 citation statements)

References 29 publications

(69 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…If this is the case, it would appear that an isomerization event, probably reflecting conformational changes involving rearrangement of the enzyme into an active form, is required for the complete reconstitution of apoenzyme. Similar rearrangements have been observed for other PLP-dependent enzymes (Churchich & Farrelly, 1969;Reed & Schnackerz, 1979;Bartholmes et al, 1980). The peculiar time-dependent spectral changes observed in the presence of D-Dopa for both mutant proteins merit comment.…”

Section: Discussionsupporting

confidence: 74%

See 1 more Smart Citation

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

et al. 1997

View full text Add to dashboard Cite

Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111 S mutant enzymes exhibit kc,, values of about 50% and 15%, respectively, at pH 6.8, while the K, values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (LJ-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-1 1 ]-+Ala mutant displays a reduced affinity for pyridoxal 5"phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pK, and pKb for the groups involved in catalysis were determined for the wild-type and the C l l l A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420 -+ 390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-1 1 1 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Bom Voltattorni C. 1991. Eur JBiochern 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation.Keywords: active site; decarboxylase; pyridoxal 5"phosphate; site-directed mutagenesis The versatility of pyridoxal 5"phosphate (PLP) as a co-factor is demonstrated by the wide variety of reactions that enzymes dependent upon it catalyze. Although detailed catalytic mechanisms and information correlating structure and function have been reported for many PLP dependent enzymes, such information is conspicuously lacking for PLP dependent decarboxylases.Even though aromatic amino acid decarboxylases have been cloned from a number of mammalian (Ichinose et al., 1989;Tanaka et al., 1989;Kang & Joh, 1990;Taketoshi et al., 1990), insect (Eveleth et al., 1986, and plant sources (DeLuca et al., 1989;Kawalleck et al., 1993; Facchini & DeLuca, 1994)

show abstract

Section: Discussionsupporting

confidence: 74%

“…For spectrophotometric measurements, PLP was added to a final concentration of 50 p M to both the apoenzyme (8 pM) and reference cuvette and spectra were taken at various times. Rapid quenching of the internal aldimine was performed as described (Bartholmes et al, 1980).…”

Section: Preparation and Reconstitution Of Apoenzymesmentioning

confidence: 99%

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

et al. 1997

View full text Add to dashboard Cite

show abstract

“…This conformational change in the bA subunit results in more efficient binding of PLP to the bB subunit [28], generating an Ecb 2 with both active sites bound to PLP. Kinetic and calorimetric studies of PLP binding to the apo-b 2 subunit of tryptophan synthase from E. coli have suggested four binding processes [29,41], which are consistent with these structural studies.…”

Section: Mechanism Of Plp Bindingsupporting

confidence: 79%

Large conformational changes in the Escherichia coli tryptophan synthase β₂ subunit upon pyridoxal 5′‐phosphate binding

et al. 2010

View full text Add to dashboard Cite

To understand the basis for the lower activity of the tryptophan synthase beta(2) subunit in comparison to the alpha(2)beta(2) complex, we determined the crystal structures of apo-beta(2) and holo-beta(2) from Escherichia coli at 3.0 and 2.9 A resolutions, respectively. To our knowledge, this is the first report of both beta(2) subunit structures with and without pyridoxal-5'-phosphate. The apo-type molecule retained a dimeric form in solution, as in the case of the holo-beta(2) subunit. The subunit structures of both the apo-beta(2) and the holo-beta(2) forms consisted of two domains, namely the N domain and the C domain. Although there were significant structural differences between the apo- and holo-structures, they could be easily superimposed with a 22 degrees rigid body rotation of the C domain. The pyridoxal-5'-phosphate-bound holo-form had multiple interactions between the two domains and a long loop (residues 260-310), which were missing in the apo-form. Comparison of the structures of holo-Ecbeta(2) and Stbeta(2) in the alpha(2)beta(2) complex from Salmonella typhimurium (Stalpha(2)beta(2)) identified the cause of the lower enzymatic activity of holo-Ecbeta(2) in comparison with Stalpha(2)beta(2). The substrate (indole) gate residues, Tyr279 and Phe280, block entry of the substrate into the beta(2) subunit, although the indole can directly access the active site as a result of a wider cleft between the N and C domains in the holo-Ecbeta(2) subunit. In addition, the structure around betaAsp305 of the holo-Ecbeta(2) subunit was similar to the open state of Stalpha(2)beta(2) with low activity, resulting in lower activity of holo-Ecbeta(2).

show abstract

“…or the L-tryptophan synthesis from indole and Lserine (Reaction 3, "B reaction")! 9 " 1 * 1, and ligand binding has been followed by different methods^1 2 " 18 ]. Covalent modification studies provided insights into the spatial relationship between the α and β active sites!…”

mentioning

confidence: 99%

Dependency on Serine Concentration of the Activity of Tryptophan Synthase. Cooperative Properties

Heilmann¹,

Bürger²

1981

Hoppe-Seyler´s Zeitschrift für physiologische Chemie

View full text Add to dashboard Cite

The activity of the enzyme trypto-L-Serine binding was found to follow the pattern phan synthase from Escherichia coli was tested as of negative cooperativity both by kinetic and by a function of the concentration of L-serine which equilibrium methods. The enzyme kinetic data serves as a substrate in the indole to tryptophan support the view that a rapid equilibration model reaction as well as for the L -serine deaminase for the enzyme · substrates complex formation is activity.not strictly obeyed. Abhängigkeit der Aktivität der Tryptophan-Synthase von der Sennkonzentration: Cooperative EigenschaftenZusammenfassung: Die Abhängigkeit der Aktivi-negative Cooperativität sowohl in kinetischen Extät des Enzyms Tryptophan-Synthase von der perimenten als auch in Gleichgewichtsdialyse-L -Serin-Konzentration wurde untersucht; L -Serin Versuchen. ist Substrat für dieses Enzym sowohl in der Tryp-Aus den enzymkinetischen Daten läßt sich erkentophansynthese aus Indol als auch in der Serinnen, daß das Enzym nicht strikt einem "rapid Desaminieruilgfcreaktion. Die Serinbindung zeigt equilibration"-Modell folgt.

show abstract

Mechanism of reconstitution of the apo.beta.2 subunit and the .alpha.2apo.beta.2 complex of tryptophan synthase with pyridoxal 5'-phosphate: kinetic studies

Cited by 27 publications

References 29 publications

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

Large conformational changes in the Escherichia coli tryptophan synthase β₂ subunit upon pyridoxal 5′‐phosphate binding

Dependency on Serine Concentration of the Activity of Tryptophan Synthase. Cooperative Properties

Contact Info

Product

Resources

About

Mechanism of reconstitution of the apo.beta.2 subunit and the .alpha.2apo.beta.2 complex of tryptophan synthase with pyridoxal 5'-phosphate: kinetic studies

Cited by 27 publications

References 29 publications

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation

Large conformational changes in the Escherichia coli tryptophan synthase β2 subunit upon pyridoxal 5′‐phosphate binding

Dependency on Serine Concentration of the Activity of Tryptophan Synthase. Cooperative Properties

Contact Info

Product

Resources

About

Large conformational changes in the Escherichia coli tryptophan synthase β₂ subunit upon pyridoxal 5′‐phosphate binding