Product binding to the alpha-carboxyl subsite results in a conformational change at the active site of O-acetylserine sulfhydrylase-A: evidence from fluorescence spectroscopy.

The intrinsic fluorescence of the pyridoxal 5'-phosphate (PLP) enzyme O-acetylserine sulfhydrylase-A (OASS-A) was studied in order to gain insight into the structural basis for binding of substrates and products and for catalysis. Excitation of OASS-A with 298-nm light gives an emission spectrum with two maxima, 337 and 498 nm. OASS-A has two tryptophan residues, and the 337-nm maximum indicates that at least one of these is exposed somewhat to aqueous solvent. The 498-nm emission observed is due to fluorescence of the PLP Schiff base. Some of this long-wavelength fluorescence is likely due to direct excitation by incident radiation. However, the concomitant quenching of 340-nm emission and the enhancement of 498-nm emission observed upon reconstitution of apoenzyme with PLP support the conclusion that some of the long-wavelength emission is due to singlet-singlet transfer from at least one tryptophan residue to the PLP Schiff base. Enhancement of 498-nm fluorescence by either of the products, acetate or cysteine, of the enzymatic reaction without a quenching of 337-nm fluorescence is consistent with triplet-singlet transfer from one or both of the tryptophan residues to the PLP Schiff base. This would require a rigid environment for the tryptophan donor when the product is bound. However, a conformational change which affected principally the environment of the PLP Schiff base, resulting in a longer lifetime of its excited singlet state, would also increase the intensity of the 498-nm emission. Enhancement of OASS-A long-wavelength fluorescence by each product requires the unprotonated form of a different group on enzyme. Enhancement by acetate binding requires the unprotonated form of an enzyme group with a pK of 7 and is insensitive to substitution on the methyl group. L-Cysteine binding enhances 498-nm fluorescence when a group with a pK of 8 is unprotonated, and substitution at the thiol or the methylene bridge does not affect the enhancement elicited. Binding of L-cysteine to free enzyme (E) likely results in the formation of the external Schiff base accompanied by a conformational change giving fluorescence enhancement. The carboxylate moiety of acetate likely binds to the alpha-carboxylate subsite for amino acid reactants such as L-cysteine, resulting in a conformational change in the internal Schiff base and giving rise to the observed fluorescence enhancement. Data are interpreted in terms of the mechanism of OASS-A.