Fluorescence of aromatic amino acids in a pyridoxal phosphate enzyme: aspartate aminotransferase
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…
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