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

Klaus D. Schnackerz, Chia Hui Tai, James W. Simmons, Tony M. Jacobson, G. S. Jagannatha Rao, Paul F. Cook

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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 Amax 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 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 α-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 hydrogenbond 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. The 31P NMR chemical shift is decreased to 4.4 ppm in the presence of L-serine, suggesting a looser binding of the S'-phosphate compared to free enzyme or the external Schiff base with L-cysteine. However, the line width in the latter case is very broad 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 -aminoacrylate intermediate results in a band with a negative Cotton effect centered at 470 nm.

Original languageEnglish (US)
Pages (from-to)12152-12160
Number of pages9
JournalBiochemistry
Volume34
Issue number38
DOIs
Publication statusPublished - Sep 1995

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Schnackerz, K. D., Tai, C. H., Simmons, J. W., Jacobson, T. M., Jagannatha Rao, G. S., & Cook, P. F. (1995). Identification and Spectral Characterization of the External Aldimine of the O-Acetylserine Sulfhydrylase Reaction. Biochemistry, 34(38), 12152-12160. https://doi.org/10.1021/bi00038a008