Abstract
The aspartate aminotransferase mutant Y70F exhibits kcat = 8% and kcat/KM = 2% of the wild type values for the transamination of aspartate and α-ketoglutarate. The affinity of the enzyme for the noncovalently bound inhibitor maleate is reduced 17-fold by the mutation, while only a 2.5-fold reduction is observed for α-methylaspartate, which forms a stable, covalent external aldimine. The high population of the quinonoid intermediate formed in the reaction of the wild type with β-hydroxyaspartate is more than 75% diminished by the mutation. The values of the Y70F Cα-H kinetic isotope effects for the aspartate reaction are larger than those of wild type (DV = 2.4 vs 1.52; D(V/K) = 2.5 vs 1.7). Conversely, the Y70F value of Q(V/K) for the glutamate reaction is decreased compared to wild type (1.75 vs 2.5). These results, combined with previous studies of Lys258 mutants, eliminate Tyr70 as an essential component of the catalytic apparatus, with the caveat that the functionality of the deleted hydroxyl group is possibly replaced by a water molecule.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 7456-7461 |
| Number of pages | 6 |
| Journal | Biochemistry |
| Volume | 30 |
| Issue number | 30 |
| DOIs | |
| State | Published - Jul 1 1991 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Tyrosine 70 Fine-Tunes the Catalytic Efficiency of Aspartate Aminotransferase. / Toney, Michael D.; Kirsch, Jack F.
In: Biochemistry, Vol. 30, No. 30, 01.07.1991, p. 7456-7461.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Tyrosine 70 Fine-Tunes the Catalytic Efficiency of Aspartate Aminotransferase
AU - Toney, Michael D.
AU - Kirsch, Jack F.
PY - 1991/7/1
Y1 - 1991/7/1
N2 - The aspartate aminotransferase mutant Y70F exhibits kcat = 8% and kcat/KM = 2% of the wild type values for the transamination of aspartate and α-ketoglutarate. The affinity of the enzyme for the noncovalently bound inhibitor maleate is reduced 17-fold by the mutation, while only a 2.5-fold reduction is observed for α-methylaspartate, which forms a stable, covalent external aldimine. The high population of the quinonoid intermediate formed in the reaction of the wild type with β-hydroxyaspartate is more than 75% diminished by the mutation. The values of the Y70F Cα-H kinetic isotope effects for the aspartate reaction are larger than those of wild type (DV = 2.4 vs 1.52; D(V/K) = 2.5 vs 1.7). Conversely, the Y70F value of Q(V/K) for the glutamate reaction is decreased compared to wild type (1.75 vs 2.5). These results, combined with previous studies of Lys258 mutants, eliminate Tyr70 as an essential component of the catalytic apparatus, with the caveat that the functionality of the deleted hydroxyl group is possibly replaced by a water molecule.
AB - The aspartate aminotransferase mutant Y70F exhibits kcat = 8% and kcat/KM = 2% of the wild type values for the transamination of aspartate and α-ketoglutarate. The affinity of the enzyme for the noncovalently bound inhibitor maleate is reduced 17-fold by the mutation, while only a 2.5-fold reduction is observed for α-methylaspartate, which forms a stable, covalent external aldimine. The high population of the quinonoid intermediate formed in the reaction of the wild type with β-hydroxyaspartate is more than 75% diminished by the mutation. The values of the Y70F Cα-H kinetic isotope effects for the aspartate reaction are larger than those of wild type (DV = 2.4 vs 1.52; D(V/K) = 2.5 vs 1.7). Conversely, the Y70F value of Q(V/K) for the glutamate reaction is decreased compared to wild type (1.75 vs 2.5). These results, combined with previous studies of Lys258 mutants, eliminate Tyr70 as an essential component of the catalytic apparatus, with the caveat that the functionality of the deleted hydroxyl group is possibly replaced by a water molecule.
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U2 - 10.1021/bi00244a013
DO - 10.1021/bi00244a013
M3 - Article
VL - 30
SP - 7456
EP - 7461
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 30
ER -