Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase

Melissa P. Hill, Elizabeth C. Carroll, Mai C. Vang, Trevor A. Addington, Michael D. Toney, Delmar S. Larsen

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)16953-16961
Number of pages9
JournalJournal of the American Chemical Society
Volume132
Issue number47
DOIs
StatePublished - Dec 1 2010

Fingerprint

Pyridoxal Phosphate
Schiff Bases
Aspartate Aminotransferases
Catalysis
Phosphates
Light
Deprotonation
Aspartic Acid
Water
Enzymes
Hot Temperature
Lighting
Isotopes
Ground state
Amino acids
Absorption spectra
Glutamic Acid
Catalyst activity
Spectrum Analysis
Spectroscopy

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Hill, M. P., Carroll, E. C., Vang, M. C., Addington, T. A., Toney, M. D., & Larsen, D. S. (2010). Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase. Journal of the American Chemical Society, 132(47), 16953-16961. https://doi.org/10.1021/ja107054x

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 journalArticle

Hill, MP, Carroll, EC, Vang, MC, Addington, TA, Toney, MD & Larsen, DS 2010, 'Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase', Journal of the American Chemical Society, vol. 132, no. 47, pp. 16953-16961. https://doi.org/10.1021/ja107054x
Hill, Melissa P. ; Carroll, Elizabeth C. ; Vang, Mai C. ; Addington, Trevor A. ; Toney, Michael D. ; Larsen, Delmar S. / Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase. In: Journal of the American Chemical Society. 2010 ; Vol. 132, No. 47. pp. 16953-16961.
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