Fluorescence of Aromatic Amino Acids in a Pyridoxal Phosphate Enzyme: Aspartate Aminotransferase

Open Access
Publisher Website
Google Scholar
Abstract
At pH 8.3, the fluorescence spectrum of apoaspartate aminotransferase is characteristic of buried tryptophans (maximum at 330 nm and width at half-height equal to 51 nm). Its quantum yield is 1.69 times larger than for tryptophan in H2O and the mean decay time is 2.5 ns for the fluorescence emitted at wavelengths higher than 335 nm. Polarization of excitation spectrum (minimum at 305 nm for an emission at 360 nm), suggests an inter-tryptophan energy transfer. Accessibility to a quencher of fluorescence indicates that 34% of the fluorescence can be extinguished by iodide with a quenching constant of 4 M−1; as shown by solvent perturbation spectroscopy, this partial accessibility is related to two tryptophan residues accessible to solvent. At pH 5, the relative quantum yield is slightly lower than at pH 8.3 (1.65). Binding of the pyridoxal-P coenzyme diminishes the fluorescence quantum yield relative to tryptophan to 0.51 at pH 8.3 and 0.595 at pH 5; the decrease is smaller in the presence of pyridoxamine-P Since the fluorescence of the coenzyme is very weak it is difficult to observe its emission sensitized by tryptophan, nevertheless, since the quenching is larger for pyridoxal-P that absorbs at 360 nm than for reduced pyridoxal-P that absorbs at 330 nm, it is deduced that the energy is transferred preferentially from exposed tryptophans. It is proposed that conformational changes in the vicinity of buried tryptophans are responsible for the remaining quenching. This hypothesis of conformational changes induced by the binding of the coenzyme is in agreement with the observed fluorescence emission of tyrosine. In the apoenzyme the tyrosine quantum yield is zero and the energy is entirely transferred to tryptophan. In the holoenzyme the quantum yield is low and the efficiency of transfer to tryptophan is 0.13 in pyridoxal-P form and 0.7 in pyridoxamine-P form. According to the Förster theory of long-range energy transfer, a change of transfer efficiency can be attributed to a modification either of the mutual orientation of tyrosine and tryptophan residues or of the distance between these residues: both interpretations correspond to a conformational change.