Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase.
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 C(α)-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 C(α)-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 C(α)-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 C(α)-H pK(a) that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.
Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States.
Melissa P
MP
Hill
Elizabeth C
EC
Carroll
Mai C
MC
Vang
Trevor A
TA
Addington
Michael D
MD
Toney
Delmar S
DS
Larsen
D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis / Q000528:radiation effects*; D007700:Kinetics; D008027:Light; D009154:Mutation; D055668:Photochemical Processes; D011522:Protons; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*; D012545:Schiff Bases / Q000737:chemistry; D012996:Solutions; D013696:Temperature
D011522:Protons; D012545:Schiff Bases; D012996:Solutions; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases