TRYPTOPHAN LUMINESCENCE AS A PROBE OF ENZYME CONFORMATION ALONG THE O-ACETYLSERINE SULFHYDRYLASE REACTION PATHWAY

O-Acetylserine sulfhydrylase A (OASS-A) is a pyridoxal 5'-phosphate- ( PLP-) dependent enzyme that catalyzes the last step in the synthesis o f L-cysteine, the beta-replacement of acetate in O-acetyl-L-serine (OA S) by sulfide. The phosphorescence properties of the two tryptophans o f wild-type OASS-A, W51 and W162, and of W162 in the W51Y mutant prote in have been characterized over the temperature range 170-273 K. In gl asses at 170 K, the apoenzyme exhibits a phosphorescence spectrum whic h is the superposition of two spectra with well-resolved 0,0 vibronic bands centered at 405 and 410 nm, the blue lambda(max) 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 t he OASS-A holoenzyme reduces the intrinsic fluorescence by 40-45%, but the spectrum is unaltered except for the appearance of the internal S chiff base ketoenamine fluorescence band centered at 484 nm. The phosp horescence is strongly quenched by PLP, with about 70% reduction in in tensity and lifetime, Further, the phosphorescence spectrum of the hol oprotein exhibits a single and narrow 0,0 vibronic band centered at 40 5 nm and a broad band in the 450-550-nm range resulting fron delayed f luorescence of the ketoenamine tautomer of the internal Schiff base, s ensitized by triplet-singlet energy transfer from tryptophan to the ke toenamine 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 b y 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 Angstrom. Substrates other th an OAS affect only the intensity of the coenzyme fluorescence band (48 4 nm) and the intensity of delayed fluorescence relative to that of ph osphorescence, effects that are attributable to changes in fluorescenc e quantum yield of the ketoenamine chromophore. Addition of GAS, on th e other hand, leads to a splitting of the 0,0 vibronic band in the pho sphorescence spectrum of W162, yielding poorly resolved peaks at 406, and 408.5 nm, indicating thereby a change in the environment of the tr yptophan residue and therefore in the conformation of the macromolecul e as the internal Schiff base is converted to the alpha-aminoacrylate Schiff base, In buffer at 273 K, both the fluorescence and phosphoresc ence spectra relax to longer wavelengths and the phosphorescence lifet ime is reduced to a few milliseconds, all indications that W162 is in a flexible region of the macromolecule, probably in close proximity to tile aqueous interface. The phosphorescence lifetime in fluid medium reveals conformational heterogeneity in OASS-A and unveils important s tructure modulating effects of cofactor, substrates, and pH. Binding o f PLP to the apoprotein increases the rigidity of the polypeptide in t he region of W162 (in agreement vith the greater thermal stability of the holoprotein), while OAS and L-serine have an opposite effect, Incr easing the pH from 6.5 to 9 results in a 1.7-fold increase in tau(av) and a change in the relative amplitudes of the two lifetime components . Since the phosphorescence originates from a single tryptophan residu e, the two tau components reflect distinct conformations of the subuni t, in this case the conformational equilibrium (slow on the phosphores cence time scale) is governed by one or more groups in the protein wit h a pK around 8.