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.
McClure
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GD
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth 76107.
Cook
P F
PF
D000085:Acetates / Q000494:pharmacology; D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000494:pharmacology; D003547:Cysteine Synthase / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012044:Regression Analysis; D012486:Salmonella typhimurium / Q000201:enzymology; D012545:Schiff Bases; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D000085:Acetates; D012545:Schiff Bases; D012694:Serine; D011732:Pyridoxal Phosphate; D014364:Tryptophan; D003547:Cysteine Synthase; C043943:O-acetylserine; D003545:Cysteine