Identification and Spectral Characterization of the External Aldimine of the O-Acetylserine Sulfhydrylase Reaction

Abstract

The O-acetylserine sulfhydrylase (OASS) reaction has been studied using a number of spectral probes including UV--visible, fluorescence, circular dichroism, and 31P 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 31P 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.(ABSTRACT TRUNCATED AT 250 WORDS)

A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex

“…1C) (8,9,31,41,42). Specifically, the formation of a hydrogen bond between Thr 69 in the active site and the ␣-carboxylate of the OAS in internal Schiff base linkage to PLP is thought to trigger a transition from an open to a closed form of the enzyme in the S. typhimurium OASS.…”

Role of Pyridoxal 5′-Phosphate in the Structural Stabilization of O-Acetylserine Sulfhydrylase

“…The 31 P NMR signal of the phosphate group of OASS-bound PLP shows an upfield shift upon exposure to 2 M GdnHCl from 5.13 to 4.54 ppm (Table I). An upfield shift was also observed after an open to close conformational transition when native OASS forms an external aldimine with L-serine (14). However, as indicated by the absorption data and by the observation that free PLP and OASS in the presence of denaturant exhibit the same peak at 4.54 ppm, the most likely explanation for the observed behavior is the breakage of the covalent bond between the PLP and the side chain of Lys-41.…”

“…In the absence of substrates or products, PLP is covalently bound via a Schiff base to the ⑀-amino group of Lys-41 (12). The internal aldimine (13) exists as an equilibrium of enolimine and ketoenamine tautomers with absorption maxima at 330 and 412 nm, respectively (8,14). The threedimensional structure of the A-isozyme of OASS from S. typhimurium (EC 4.2.99.8) has been recently determined at 2.2 Å resolution (5,15), and the catalytic competence of the enzyme in the crystalline state has been assessed by polarized absorption microspectrophotometry (16).…”

“…A large conformational change takes place upon ligand binding (14,15,19), indicating that some regions of the protein are quite flexible. The comparison of the emission properties of apo-and holoenzyme indicates the presence of an energy transfer between tryptophans and the coenzyme (18,21).…”

Molecular Basis of Cysteine Biosynthesis in Plants

“…1C, step 4). The active site lysine reacts with this intermediate, releasing cysteine and regenerating the Schiff base (20,21). Site-directed mutagenesis of Salmonella typhimurium OASS (StOASS) confirmed the importance of the lysine (22) and the contribution of an active site serine to stabilizing the pyridoxal ring in the reaction (23).…”