Anaerobiospirillum succiniciproducens Phosphoenolpyruvate Carboxykinase: Mutagenesis at Metal Site 2

Ana María Jabalquinto, Maris Laivenieks, Fernando D. González-Nilo, María Victoria Encinas, Gregory Zeikus, Emilio Cardemil

Resultado de la investigación: Article

4 Citas (Scopus)

Resumen

Phosphoenolpyruvate (PEP) carboxykinases harbor two divalent metal-binding sites. One cation interacts with the enzyme (metal binding site 1) to elicit activation, while a second cation (metal binding site 2) interacts with the nucleotide to serve as the metal nucleotide substrate. Mutants of Anaerobiospirillum succiniciproducens PEP carboxykinase have been constructed where Thr249 and Asp262, two residues of metal binding site 2 of the enzyme, were altered. Binding of the 3′(2′ )-O-(N-methylantraniloyl) derivative of ADP provides a test of the structural integrity of these mutants. The conservative mutation (Asp262Glu) retains a significant proportion of the wild type enzymatic activity. Meanwhile, removal of the OH group of Thr249 in the Thr249Ala mutant causes a decrease in Vmax by a factor of 1.1 × 104. Molecular modeling of wild type and mutant enzymes suggests that the lower catalytic efficiency of the Thr249Ala enzyme could be explained by a movement of the lateral chain of Lys248, a critical catalytic residue, away from the reaction center.

Idioma originalEnglish
Páginas (desde-hasta)515-519
Número de páginas5
PublicaciónJournal of Protein Chemistry
Volumen22
N.º6
DOI
EstadoPublished - 1 ago 2003

Huella dactilar

Anaerobiospirillum
Mutagenesis
Phosphoenolpyruvate
Binding sites
Metals
Enzymes
Binding Sites
Nucleotides
Cations
Positive ions
Administrative data processing
Molecular modeling
Structural integrity
Ports and harbors
Adenosine Diphosphate
Chemical activation
Derivatives
Mutation
Substrates

ASJC Scopus subject areas

  • Biochemistry

Citar esto

Jabalquinto, Ana María ; Laivenieks, Maris ; González-Nilo, Fernando D. ; Encinas, María Victoria ; Zeikus, Gregory ; Cardemil, Emilio. / Anaerobiospirillum succiniciproducens Phosphoenolpyruvate Carboxykinase : Mutagenesis at Metal Site 2. En: Journal of Protein Chemistry. 2003 ; Vol. 22, N.º 6. pp. 515-519.
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title = "Anaerobiospirillum succiniciproducens Phosphoenolpyruvate Carboxykinase: Mutagenesis at Metal Site 2",
abstract = "Phosphoenolpyruvate (PEP) carboxykinases harbor two divalent metal-binding sites. One cation interacts with the enzyme (metal binding site 1) to elicit activation, while a second cation (metal binding site 2) interacts with the nucleotide to serve as the metal nucleotide substrate. Mutants of Anaerobiospirillum succiniciproducens PEP carboxykinase have been constructed where Thr249 and Asp262, two residues of metal binding site 2 of the enzyme, were altered. Binding of the 3′(2′ )-O-(N-methylantraniloyl) derivative of ADP provides a test of the structural integrity of these mutants. The conservative mutation (Asp262Glu) retains a significant proportion of the wild type enzymatic activity. Meanwhile, removal of the OH group of Thr249 in the Thr249Ala mutant causes a decrease in Vmax by a factor of 1.1 × 104. Molecular modeling of wild type and mutant enzymes suggests that the lower catalytic efficiency of the Thr249Ala enzyme could be explained by a movement of the lateral chain of Lys248, a critical catalytic residue, away from the reaction center.",
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Anaerobiospirillum succiniciproducens Phosphoenolpyruvate Carboxykinase : Mutagenesis at Metal Site 2. / Jabalquinto, Ana María; Laivenieks, Maris; González-Nilo, Fernando D.; Encinas, María Victoria; Zeikus, Gregory; Cardemil, Emilio.

En: Journal of Protein Chemistry, Vol. 22, N.º 6, 01.08.2003, p. 515-519.

Resultado de la investigación: Article

TY - JOUR

T1 - Anaerobiospirillum succiniciproducens Phosphoenolpyruvate Carboxykinase

T2 - Mutagenesis at Metal Site 2

AU - Jabalquinto, Ana María

AU - Laivenieks, Maris

AU - González-Nilo, Fernando D.

AU - Encinas, María Victoria

AU - Zeikus, Gregory

AU - Cardemil, Emilio

PY - 2003/8/1

Y1 - 2003/8/1

N2 - Phosphoenolpyruvate (PEP) carboxykinases harbor two divalent metal-binding sites. One cation interacts with the enzyme (metal binding site 1) to elicit activation, while a second cation (metal binding site 2) interacts with the nucleotide to serve as the metal nucleotide substrate. Mutants of Anaerobiospirillum succiniciproducens PEP carboxykinase have been constructed where Thr249 and Asp262, two residues of metal binding site 2 of the enzyme, were altered. Binding of the 3′(2′ )-O-(N-methylantraniloyl) derivative of ADP provides a test of the structural integrity of these mutants. The conservative mutation (Asp262Glu) retains a significant proportion of the wild type enzymatic activity. Meanwhile, removal of the OH group of Thr249 in the Thr249Ala mutant causes a decrease in Vmax by a factor of 1.1 × 104. Molecular modeling of wild type and mutant enzymes suggests that the lower catalytic efficiency of the Thr249Ala enzyme could be explained by a movement of the lateral chain of Lys248, a critical catalytic residue, away from the reaction center.

AB - Phosphoenolpyruvate (PEP) carboxykinases harbor two divalent metal-binding sites. One cation interacts with the enzyme (metal binding site 1) to elicit activation, while a second cation (metal binding site 2) interacts with the nucleotide to serve as the metal nucleotide substrate. Mutants of Anaerobiospirillum succiniciproducens PEP carboxykinase have been constructed where Thr249 and Asp262, two residues of metal binding site 2 of the enzyme, were altered. Binding of the 3′(2′ )-O-(N-methylantraniloyl) derivative of ADP provides a test of the structural integrity of these mutants. The conservative mutation (Asp262Glu) retains a significant proportion of the wild type enzymatic activity. Meanwhile, removal of the OH group of Thr249 in the Thr249Ala mutant causes a decrease in Vmax by a factor of 1.1 × 104. Molecular modeling of wild type and mutant enzymes suggests that the lower catalytic efficiency of the Thr249Ala enzyme could be explained by a movement of the lateral chain of Lys248, a critical catalytic residue, away from the reaction center.

KW - Anaerobiospirillum succiniciproducens

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KW - Site-directed mutagenesis

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