The K258R mutant of aspartate aminotransferase stabilizes the quinonoid intermediate

Michael D. Toney, Jack F. Kirsch

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Lys-258 of aspartate aminotransferase forms a Schiff base with pyridoxal phosphate and is responsible for catalysis of the 1,3-prototropic shift central to the transamination reaction sequence. Substitution of arginine for Lys-258 stabilizes the otherwise elusive quinonoid intermediate, as assessed by the long wavelength absorption bands observed in the reactions of this mutant with several amino acid substrates. The external aldimine intermediate is not detectable during reactions of this mutant with amino acids, although the inhibitor α-methylaspartate does slowly and stably form this species. These results suggest that external aldimine formation is one of the rate-determining steps of the reaction. The pyridoxamine-5′-phosphate-like enzyme form (330-nm absorption maximum) is unreactive toward keto acid substrates, and the coenzyme bound to this species is not dissociable from the protein.

Original languageEnglish (US)
Pages (from-to)23900-23903
Number of pages4
JournalJournal of Biological Chemistry
Volume266
Issue number35
StatePublished - Dec 15 1991

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Aspartate Aminotransferases
Pyridoxamine
Keto Acids
Amino Acids
Pyridoxal Phosphate
Schiff Bases
Coenzymes
Substrates
Catalysis
Arginine
Absorption spectra
Substitution reactions
Phosphates
Wavelength
Enzymes
Proteins
pyridoxamine phosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

The K258R mutant of aspartate aminotransferase stabilizes the quinonoid intermediate. / Toney, Michael D.; Kirsch, Jack F.

In: Journal of Biological Chemistry, Vol. 266, No. 35, 15.12.1991, p. 23900-23903.

Research output: Contribution to journalArticle

Toney, Michael D. ; Kirsch, Jack F. / The K258R mutant of aspartate aminotransferase stabilizes the quinonoid intermediate. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 35. pp. 23900-23903.
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abstract = "Lys-258 of aspartate aminotransferase forms a Schiff base with pyridoxal phosphate and is responsible for catalysis of the 1,3-prototropic shift central to the transamination reaction sequence. Substitution of arginine for Lys-258 stabilizes the otherwise elusive quinonoid intermediate, as assessed by the long wavelength absorption bands observed in the reactions of this mutant with several amino acid substrates. The external aldimine intermediate is not detectable during reactions of this mutant with amino acids, although the inhibitor α-methylaspartate does slowly and stably form this species. These results suggest that external aldimine formation is one of the rate-determining steps of the reaction. The pyridoxamine-5′-phosphate-like enzyme form (330-nm absorption maximum) is unreactive toward keto acid substrates, and the coenzyme bound to this species is not dissociable from the protein.",
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