Light-Enhanced Catalysis by Pyridoxal Phosphate-Dependent Aspartate Aminotransferase

Abstract

The mechanisms of pyridoxal 5′-phosphate (PLP) dependent enzymes require substrates to form covalent “external aldimine” intermediates, which absorb light strongly between 410 nm 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, 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 based on 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 58,600-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 Cα-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.

Carbon Acidity in Enzyme Active Sites

“…Previous studies with aspartate aminotransferase defined rate constants for a mechanism in which the central 1,3-prototropic shift occurs as a concerted double proton transfer, avoiding the carbanionic quinonoid intermediate (Goldberg and Kirsch, 1996). A more recent study proved the existence of the quinonoid intermediate on the productive pathway (Hill et al, 2010). Therefore, a FEP including the quinonoid intermediate on the reaction pathway was determined by global optimization.…”

Aspartate aminotransferase: An old dog teaches new tricks

“…[86] When AAT is irradiated with 250 mW of 440 nm light, the value of k cat is increased 2.3-fold (Figure 9). A photochemical mechanism similar to that for Schiff bases in water was found for AAT, wherein the excited singlet state undergoes intersystem crossing to a triplet state that is responsible for accelerated loss of the proton from Cα to give the carbanionic intermediate.…”

The Effect of Visible Light on the Catalytic Activity of PLP‐Dependent Enzymes

“…[28] Interestingly, when bound to the active site lysine of an aspartate amine transaminase, blue light pulses actually supported the formation of the carbanionic intermediate within microseconds, resulting in an increased catalytic activity. [29][30][31] A similar photoactivation has already been described for the enzymatic decarboxylation of 5hydroxytryptophan. [32] Although some studies exist, to the best of our knowledge, the effect of light on the stability of PLPdependent enzymes has not been in scientific focus in the first place.…”

“…As PLP absorbs specifically in the blue light region of the visible light spectrum, around 440 nm, it has been reported, that PLP can be stimulated by blue light illumination. [28,29] To determine changes in the spectrum of free PLP during illumination in different setups, 0.1 mM PLP solutions in HEPES buffer (pH 7.5) were subjected to illumination by a single blue LED (~60 mW/ cm 2 ) or by ordinary laboratory and sunlight (~0.25 mW/cm 2 ; Figure 2A). In the following, light conditions consisting of laboratory and sunlight are referred to as "normal light conditions".…”