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

Ahmed, Salman; McPhie, Peter; Miles, Edith Wilson

doi:10.1074/jbc.271.15.8612

Cited by 18 publications

(22 citation statements)

References 26 publications

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“…The structure could be either a neutral form (structure IIa), resulting from dissociation of Ha and transfer of Hb to the phenolic hydroxyl, or a dipolar ionic form (structure IIb), resulting from dissociation of Hb. The observed max ϭ 334 nm is consistent with that of the neutral enolimine form of the Schiff base, which is in equilibrium with the resonance-stabilized ketoenamine form in the wild type tryptophan synthase (35). The enolimine form predominates following a thermally induced reversible conformational transition of the ␤ 2 subunit (35).…”

Section: Table Isupporting

confidence: 66%

“…The observed max ϭ 334 nm is consistent with that of the neutral enolimine form of the Schiff base, which is in equilibrium with the resonance-stabilized ketoenamine form in the wild type tryptophan synthase (35). The enolimine form predominates following a thermally induced reversible conformational transition of the ␤ 2 subunit (35). The high pH form of tryptophanase also has max ϭ 337 nm, consistent with an enolimine structure (36 -38).…”

Section: Table Isupporting

confidence: 59%

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Mutation of an Active Site Residue of Tryptophan Synthase (β-Serine 377) Alters Cofactor Chemistry

Jhee

Yang²,

Ahmed³

et al. 1998

Journal of Biological Chemistry

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To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (␤-Ser 377 ) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pK a ϳ ϳ 7.7) from a form with a protonated internal aldimine nitrogen ( max ‫؍‬ 416 nm) to a deprotonated form ( max ‫؍‬ 336 nm), whereas the absorption spectra of the wild type tryptophan synthase ␤ 2 subunit and ␣ 2 ␤ 2 complex are pHindependent. The reaction of the S377D ␣ 2 ␤ 2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands ( max ‫؍‬ 498 -510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pK a of the internal aldimine nitrogen and promoting formation of quinonoid intermediates.An important question in investigations of enzyme structure and function is how enzymes have evolved different reaction and substrate specificities. Pyridoxal phosphate (PLP) 1 -dependent enzymes are attractive targets for addressing this question because they catalyze a wide variety of reactions of amino acids (1, 2). The enzyme protein directs and restricts the catalytic potential of the bound PLP to provide the substrate and reaction specificity and the enhanced reaction rate of the enzyme (3, 4). Thus, it is important to understand how the protein structure controls the cofactor chemistry and enhances catalytic rates.Information on how the protein structure controls PLP-dependent reactions is beginning to emerge from x-ray crystallography and from sequence comparisons designed to establish evolutionary relationships (5, 6). One of the most important and best studied interactions between the cofactor and the protein active site of all PLP enzymes is that between the pyridine nitrogen of PLP and an amino acid side chain (see Fig.

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Section: Table Isupporting

confidence: 66%

Section: Table Isupporting

confidence: 59%

Mutation of an Active Site Residue of Tryptophan Synthase (β-Serine 377) Alters Cofactor Chemistry

Jhee

Yang²,

Ahmed³

et al. 1998

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Such an isosbestic point suggests an interconversion between two chemical species during ablation (15). Our analysis suggests that this is not a result of the generation of oxidized methemoglobin, as the 550 nm isosbestic point does not correlate with published isosbestic values for methemoglobin (16).…”

Section: Discussionmentioning

confidence: 55%

Liver Tumor Gross Margin Identification and Ablation Monitoring during Liver Radiofrequency Treatment

Hsu

Razavi

et al. 2005

Journal of Vascular and Interventional Radiology

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“…1B). With other PLP‐dependent enzymes, the 420 nm‐absorption species is generally attributed to the ketoenamine form of the internal aldimine (Ahmed et al 1996; Li et al 1997; Ikushiro et al 1998; Zhou and Toney 1999) (Fig. 1C), but the assignment of the 330 nm‐absorption species is less straightforward, as several potential structures have the same absorption maximum.…”

Section: Resultsmentioning

confidence: 99%

Conversion of 5‐aminolevulinate synthase into a more active enzyme by linking the two subunits: Spectroscopic and kinetic properties

2005

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The two active sites of dimeric 5-aminolevulinate synthase (ALAS), a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme, are located on the subunit interface with contribution of essential amino acids from each subunit. Linking the two subunits into a single polypeptide chain dimer (2XALAS) yielded an enzyme with an approximate sevenfold greater turnover number than that of wild-type ALAS. Spectroscopic and kinetic properties of 2XALAS were investigated to explore the differences in the coenzyme structure and kinetic mechanism relative to those of wild-type ALAS that confer a more active enzyme. The absorption spectra of both ALAS and 2XALAS had maxima at 410 and 330 nm, with a greater A 410 /A 330 ratio at pH ∼7.5 for 2XALAS. The 330 nm absorption band showed an intense fluorescence at 385 nm but not at 510 nm, indicating that the 330 nm absorption species is the substituted aldamine rather than the enolimine form of the Schiff base. The 385 nm emission intensity increased with increasing pH with a single pK of ∼8.5 for both enzymes, and thus the 410 and 330 nm absorption species were attributed to the ketoenamine and substituted aldamine, respectively. Transient kinetic analysis of the formation and decay of the quinonoid intermediate EQ 2 indicated that, although their rates were similar in ALAS and 2XALAS, accumulation of this intermediate was greater in the 2XALAS-catalyzed reaction. Collectively, these results suggest that ketoenamine is the active form of the coenzyme and forms a more prominent coenzyme structure in 2XALAS than in ALAS at pH ∼7.5.

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

Cited by 18 publications

References 26 publications

Mutation of an Active Site Residue of Tryptophan Synthase (β-Serine 377) Alters Cofactor Chemistry

Mutation of an Active Site Residue of Tryptophan Synthase (β-Serine 377) Alters Cofactor Chemistry

Liver Tumor Gross Margin Identification and Ablation Monitoring during Liver Radiofrequency Treatment

Conversion of 5‐aminolevulinate synthase into a more active enzyme by linking the two subunits: Spectroscopic and kinetic properties

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