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 alpha-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine C-alpha-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-alpha-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-alpha-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-alpha-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-alpha-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.