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 language | English (US) |
|---|---|
| Pages (from-to) | 23900-23903 |
| Number of pages | 4 |
| Journal | Journal of Biological Chemistry |
| Volume | 266 |
| Issue number | 35 |
| State | Published - Dec 15 1991 |
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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 journal › Article
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TY - JOUR
T1 - The K258R mutant of aspartate aminotransferase stabilizes the quinonoid intermediate
AU - Toney, Michael D.
AU - Kirsch, Jack F.
PY - 1991/12/15
Y1 - 1991/12/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0026343177&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0026343177&partnerID=8YFLogxK
M3 - Article
C2 - 1748661
AN - SCOPUS:0026343177
VL - 266
SP - 23900
EP - 23903
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 35
ER -