The O-acetylserine sulfhydrylase (OASS) reaction has been studied using a number of spectral probes including UV-visible, fluorescence, circular dichroism, and P-31 NMR spectroscopy. The addition of L-cysteine, L-alanine, and glycine to OASS results in a shift in lambda(max) of 412 nm for the internal Schiff base to 418 nm resulting from the formation of the external Schiff base. The addition of L-serine or O-methyl-D,L-serine gives decreases of the absorbance of unliganded enzyme at 412 nm of about 50% and 20%, respectively, concomitant with an increase in the absorbance at 320 nm and a shift in the lambda(max) of the remaining visible absorbance to 418 nm. The spectral shifts observed in the presence of L-serine are suggestive of establishing an equilibrium between different forms of external Schiff base. The concentration dependence of the changes at 440 (L-cysteine) and 320 nm (L-serine) provides an estimate of the dissociation constant for the external aldimine, The pH dependence of the dissociation constant suggests the alpha-amine of the amino acid must be unprotonated for nucleophilic attack at C4' of PLP, and an enzyme side chain must be unprotonated to hydrogen-bond the thiol or hydroxyl side chain of the amino acid. When L-cysteine is the amino acid, the thiol side chain must be protonated to hydrogen-bond to the unprotonated enzyme side chain. The P-31 NMR chemical shift is increased from 5.2 ppm for unliganded enzyme to 5.3 ppm in the presence of L-cysteine, signaling a tighter interaction at the 5'-phosphate upon formation of the external Schiff base, The P-31 NMR chemical shift is decreased to 4.4 ppm in the presence of L-serine, suggesting a looser binding of the 5'-phosphate compared to free enzyme or the external Schiff base with L-cysteine. However, the line width in the latter case is very bread at about 50 Hz, suggesting the presence of more than one species. The spectrum in the presence of L-serine gives a change in the chemical shift to higher field as the temperature decreases. Addition of L-cysteine or L-serine significantly changes the far-UV CD spectrum, likely reflecting the closing of the active site. In addition, an induced dichroism of the PLP cofactor is observed in the visible region of the CD spectrum. Free enzyme gives a positive Cotton effect at 412 nm, while in the presence of L-cysteine or glycine the positive Cotton effect shifts to 418 nm with the same intensity as that observed for free enzyme, The visible CD in the presence of L-serine exhibits positive Cotton effects at 320 and 418 nm, while formation of the alpha-aminoacrylate intermediate results in a band with a negative Cotton effect centered at 470 nm.
