title: 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)
authors.affiliation: Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center at Fort Worth 76107-2699, USA.
authors.forename: K D
authors.initials: KD
authors.lastname: Schnackerz
authors.forename: C H
authors.initials: CH
authors.lastname: Tai
authors.forename: J W
authors.initials: JW
authors.lastname: Simmons
authors.forename: T M
authors.initials: TM
authors.lastname: Jacobson
authors.forename: G S
authors.initials: GS
authors.lastname: Rao
authors.forename: P F
authors.initials: PF
authors.lastname: Cook
mesh_terms: D000596:Amino Acids / Q000494:pharmacology; D002942:Circular Dichroism; D003547:Cysteine Synthase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007097:Imines; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D010759:Phosphorus Isotopes; D012486:Salmonella typhimurium / Q000201:enzymology; D012545:Schiff Bases; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
chemical_list: D000596:Amino Acids; D007097:Imines; D010759:Phosphorus Isotopes; D012545:Schiff Bases; C095928:O-methylserine; D012694:Serine; D003547:Cysteine Synthase