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 Scopus citations

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

ASJC Scopus subject areas

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

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    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