Abstract
The mechanisms of pyridoxal 5′-phosphate (PLP)-dependent enzymes require substrates to form covalent "external aldimine" intermediates, which absorb light strongly between 410 and 430 nm. Aspartate aminotransferase (AAT) is a prototypical PLP-dependent enzyme that catalyzes the reversible interconversion of aspartate and α-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine Cα-H bond to give a carbanionic quinonoid intermediate is partially rate limiting in the thermal AAT reaction. We show that excitation of the 430-nm external aldimine absorption band increases the steady-state catalytic activity of AAT, which is attributed to the photoenhancement of Cα-H deprotonation on the basis of studies with Schiff bases in solution. Blue light (250 mW) illumination gives an observed 2.3-fold rate enhancement for WT AAT activity, a 530-fold enhancement for the inactive K258A mutant, and a 58600-fold enhancement for the PLP-Asp Schiff base in water. These different levels of enhancement correlate with the intrinsic reactivities of the Cα-H bond in the different environments, with the less reactive Schiff bases exhibiting greater enhancement. Time-resolved spectroscopy, ranging from femtoseconds to minutes, was used to investigate the nature of the photoactivation of C α-H bond cleavage in PLP-amino acid Schiff bases both in water and bound to AAT. Unlike the thermal pathway, the photoactivation pathway involves a triplet state with a Cα-H pKa that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 16953-16961 |
| Number of pages | 9 |
| Journal | Journal of the American Chemical Society |
| Volume | 132 |
| Issue number | 47 |
| DOIs | |
| State | Published - Dec 1 2010 |
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ASJC Scopus subject areas
- Chemistry(all)
- Catalysis
- Biochemistry
- Colloid and Surface Chemistry
Cite this
Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase. / Hill, Melissa P.; Carroll, Elizabeth C.; Vang, Mai C.; Addington, Trevor A.; Toney, Michael D.; Larsen, Delmar S.
In: Journal of the American Chemical Society, Vol. 132, No. 47, 01.12.2010, p. 16953-16961.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase
AU - Hill, Melissa P.
AU - Carroll, Elizabeth C.
AU - Vang, Mai C.
AU - Addington, Trevor A.
AU - Toney, Michael D.
AU - Larsen, Delmar S.
PY - 2010/12/1
Y1 - 2010/12/1
N2 - The mechanisms of pyridoxal 5′-phosphate (PLP)-dependent enzymes require substrates to form covalent "external aldimine" intermediates, which absorb light strongly between 410 and 430 nm. Aspartate aminotransferase (AAT) is a prototypical PLP-dependent enzyme that catalyzes the reversible interconversion of aspartate and α-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine Cα-H bond to give a carbanionic quinonoid intermediate is partially rate limiting in the thermal AAT reaction. We show that excitation of the 430-nm external aldimine absorption band increases the steady-state catalytic activity of AAT, which is attributed to the photoenhancement of Cα-H deprotonation on the basis of studies with Schiff bases in solution. Blue light (250 mW) illumination gives an observed 2.3-fold rate enhancement for WT AAT activity, a 530-fold enhancement for the inactive K258A mutant, and a 58600-fold enhancement for the PLP-Asp Schiff base in water. These different levels of enhancement correlate with the intrinsic reactivities of the Cα-H bond in the different environments, with the less reactive Schiff bases exhibiting greater enhancement. Time-resolved spectroscopy, ranging from femtoseconds to minutes, was used to investigate the nature of the photoactivation of C α-H bond cleavage in PLP-amino acid Schiff bases both in water and bound to AAT. Unlike the thermal pathway, the photoactivation pathway involves a triplet state with a Cα-H pKa that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.
AB - The mechanisms of pyridoxal 5′-phosphate (PLP)-dependent enzymes require substrates to form covalent "external aldimine" intermediates, which absorb light strongly between 410 and 430 nm. Aspartate aminotransferase (AAT) is a prototypical PLP-dependent enzyme that catalyzes the reversible interconversion of aspartate and α-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine Cα-H bond to give a carbanionic quinonoid intermediate is partially rate limiting in the thermal AAT reaction. We show that excitation of the 430-nm external aldimine absorption band increases the steady-state catalytic activity of AAT, which is attributed to the photoenhancement of Cα-H deprotonation on the basis of studies with Schiff bases in solution. Blue light (250 mW) illumination gives an observed 2.3-fold rate enhancement for WT AAT activity, a 530-fold enhancement for the inactive K258A mutant, and a 58600-fold enhancement for the PLP-Asp Schiff base in water. These different levels of enhancement correlate with the intrinsic reactivities of the Cα-H bond in the different environments, with the less reactive Schiff bases exhibiting greater enhancement. Time-resolved spectroscopy, ranging from femtoseconds to minutes, was used to investigate the nature of the photoactivation of C α-H bond cleavage in PLP-amino acid Schiff bases both in water and bound to AAT. Unlike the thermal pathway, the photoactivation pathway involves a triplet state with a Cα-H pKa that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.
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U2 - 10.1021/ja107054x
DO - 10.1021/ja107054x
M3 - Article
C2 - 21058708
AN - SCOPUS:78649494486
VL - 132
SP - 16953
EP - 16961
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
SN - 0002-7863
IS - 47
ER -