Istituto di Biofisica     
Strambini G. B., Cioni P., Cook P. F. Tryptophan Luminescence as a Probe of Enzyme Conformation along the O-Acetylserine Sulfhydrylase Reaction Pathway. In: Biochemistry, vol. 35 (25) pp. 8392 - 8400. American Chemical Society, 1996.
O-Acetylserine sulfhydrylase A (OASS-A) is a pyridoxal 5'-phosphate- (PLP-) dependent enzyme that catalyzes the last step in the synthesis of l-cysteine, the β-replacement of acetate in O-acetyl-l-serine (OAS) by sulfide. The phosphorescence properties of the two tryptophans of wild-type OASS-A, W51 and W162, and of W162 in the W51Y mutant protein have been characterized over the temperature range 170−273 K. In glasses at 170 K, the apoenzyme exhibits a phosphorescence spectrum which is the superposition of two spectra with well-resolved 0,0 vibronic bands centered at 405 and 410 nm, the blue λmax suggesting that one of the two Trp residues in OASS-A is in a polar pocket, while the other is in a relatively hydrophobic pocket. The presence of PLP in the OASS-A holoenzyme reduces the intrinsic fluorescence by 40−45%, but the spectrum is unaltered except for the appearance of the internal Schiff base ketoenamine fluorescence band centered at 484 nm. The phosphorescence is strongly quenched by PLP, with about 70% reduction in intensity and lifetime. Further, the phosphorescence spectrum of the holoprotein exhibits a single and narrow 0,0 vibronic band centered at 405 nm and a broad band in the 450−550-nm range resulting from delayed fluorescence of the ketoenamine tautomer of the internal Schiff base, sensitized by triplet−singlet energy transfer from tryptophan to the ketoenamine tautomer of PLP. Comparison with data obtained for the W51Y mutant strongly suggests that the 405-nm phosphorescence band derives from W162, and that W51 in the wild type is entirely quenched either by singlet or triplet energy transfer to PLP or by some local group in the protein. From the rate of energy transfer, the separation between W162 and PLP is estimated to be about 25 . Substrates other than OAS affect only the intensity of the coenzyme fluorescence band (484 nm) and the intensity of delayed fluorescence relative to that of phosphorescence, effects that are attributable to changes in fluorescence quantum yield of the ketoenamine chromophore. Addition of OAS, on the other hand, leads to a splitting of the 0,0 vibronic band in the phosphorescence spectrum of W162, yielding poorly resolved peaks at 406 and 408.5 nm, indicating thereby a change in the environment of the tryptophan residue and therefore in the conformation of the macromolecule as the internal Schiff base is converted to the α-aminoacrylate Schiff base. In buffer at 273 K, both the fluorescence and phosphorescence spectra relax to longer wavelengths and the phosphorescence lifetime is reduced to a few milliseconds, all indications that W162 is in a flexible region of the macromolecule, probably in close proximity to the aqueous interface. The phosphorescence lifetime in fluid medium reveals conformational heterogeneity in OASS-A and unveils important structure modulating effects of cofactor, substrates, and pH. Binding of PLP to the apoprotein increases the rigidity of the polypeptide in the region of W162 (in agreement with the greater thermal stability of the holoprotein), while OAS and l-serine have an opposite effect. Increasing the pH from 6.5 to 9 results in a 1.7-fold increase in τav and a change in the relative amplitudes of the two lifetime components. Since the phosphorescence originates from a single tryptophan residue, the two τ components reflect distinct conformations of the subunit. In this case the conformational equilibrium (slow on the phosphorescence time scale) is governed by one or more groups in the protein with a pK around 8.
DOI: 10.1021/bi952919e
Subject O-Acetylserine sulfhydrylase A
Pyridoxal 5'phosphate-
Mutant proteins
Energy transfer

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