Activation of aposerine transhydroxymethylase by pyridoxal-5′-phosphate monomethyl Ester

doi:10.1016/S0006-291X(78)80016-3

Biochemical and Biophysical Research Communications

Volume 85, Issue 1, 14 November 1978, Pages 99-106

https://doi.org/10.1016/S0006-291X(78)80016-3 Get rights and content

Summary

Aposerine transhydroxymethylase was observed to be completely reactivated by pyridoxal-P monomethyl ester. A kinetic study showed the reactivation process to be first order with respect to apoenzyme and the coenzyme analog. Reisolation of the cofactor from the holoenzyme and its identification by thin-layer and column chromatography revealed that pyridoxal-P monomethyl ester is the active coenzyme. Spectral studies indicated that enzyme reactivated with analog forms normal enzyme-substrate complexes with glycine. We conclude from these experiments that serine transhydroxymethylase can function normally with the phosphate group of the coenzyme in its monoanionic form.

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Cited by (9)

<sup>31</sup>P NMR studies of O-acetylserine sulfhydrylase-B from Salmonella typhimurium
2009, Archives of Biochemistry and Biophysics
Citation Excerpt :
Considering the tight link between electronic and conformational states, it is not surprising that 31P NMR methods have been exploited for PLP-dependent enzyme to reveal the conformational landscape of catalytic actions by focusing on the binding modes of the phosphate group on PLP [9–14]. Resolution of the PLP cofactor and its reconstitution with the apoenzyme has been used to study the binding site of PLP for several PLP-dependent enzymes, including the A-isozyme of OASS [15–19]. Preparation of apo OASS-A by resolution of PLP was shown to be crucial for the studies of enzyme stability and dynamics [20–23].
O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine and bisulfide to l-cysteine and acetate in bacteria and higher plants. Enteric bacteria have two isozymes of OASS, A and B, produced under aerobic and anaerobic growth conditions, respectively, with different substrate specificities. The ³¹P chemical shift of the internal and external Schiff bases of PLP in OASS-B are further downfield compared to OASS-A, suggesting a tighter binding of the cofactor in the B-isozyme. The chemical shift of the internal Schiff base (ISB) of OASS-B is 6.2 ppm, the highest value reported for the ISB of a PLP-dependent enzyme. Considering the similarity in the binding sites of the PLP cofactor for both isozymes, torsional strain of the C5–C5′ bond (O4′–C5′–C5–C4) of the Schiff base is proposed to contribute to the further downfield shift. The chemical shift of the lanthionine external Schiff base (ESB) of OASS-B is 6.0 ppm, upfield from that of unliganded OASS-B, while that of serine ESB is 6.3 ppm. Changes in chemical shift suggest the torsional strain of PLP changes as the reaction proceeds.
The apoenzyme of OASS-B was prepared using hydroxylamine as the resolving reagent. Apoenzyme was reconstituted to holoenzyme by addition of PLP. Reconstitution is pseudo-first order and exhibits a final maximum recovery of 81.4%. The apoenzyme shows no visible absorbance, while the reconstituted enzyme has a UV–visible spectrum that is nearly identical to that of the holoenzyme. Steady-state fluorescence spectra gave tryptophan emission of the apoenzyme that is 3.3-fold higher than the emission of either the native or reconstituted enzyme, suggesting that PLP is a potent quencher of tryptophan emission.
Substitution of pyridoxal 5'-phosphate in D-serine dehydratase from Escherichia coli by cofactor analogues provides information on cofactor binding and catalysis
1999, Journal of Biological Chemistry
d-Serine dehydratase (DSD) is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the conversion of d-serine to pyruvate and ammonia. Spectral studies of enzyme species where the natural cofactor was substituted by pyridoxal 5′-sulfate (PLS), pyridoxal 5-deoxymethylene phosphonate (PDMP), and pyridoxal 5′-phosphate monomethyl ester (PLPMe) were used to gain insight into the structural basis for binding of cofactor and substrate analogues. PDMP-DSD exhibits 35% of the activity of the native enzyme, whereas PLS-DSD and PLPMe-DSD are catalytically inactive. The emission spectrum of native DSD when excited at 280 nm shows maxima at 335 and 530 nm. The energy transfer band at 530 nm is very likely generated as a result of the proximity of Trp-197 to the protonated internal Schiff base. The cofactor analogue-reconstituted DSD species exhibit emission intensities decreasing from PLS-DSD, to PLPMe-DSD, and PDMP-DSD, when excited at 415 nm. Large increases in fluorescence intensity at 530 (540) nm can be observed for cofactor analogue-reconstituted DSD in the presence of substrate analogues when excited at 415 nm. In the absence and presence of substrate analogues, virtually identical far UV CD spectra were obtained for all DSD species. The visible CD spectra of native DSD, PDMP-DSD, and PLS-DSD exhibit a band centered on the visible absorption maximum with nearly identical intensity. Addition of substrate analogues to native and cofactor analogue-reconstituted DSD species results in most cases in a decrease or elimination of ellipticity. The results are interpreted in terms of local conformational changes and/or changes in the orientation of the bound cofactor (analogue).
Resolution of pyridoxal 5′-phosphate from O-acetylserine sulfhydrylase from Salmonella typhimurium and reconstitution of apoenzyme with cofactor and cofactor analogues as a probe of the cofactor binding site
1995, Archives of Biochemistry and Biophysics
A procedure has been developed to prepare the apoenzyme ofO-acetylserine sulfhydrylase (apoOASS) by first converting the native enzyme to the α-aminoacrylate intermediate and dialyzing against 5Mguanidinium chloride. Aposulfhydrylase is stable for at least a month in buffers containing phosphate or phosphate analogues. Reconstitution of aposulfhydrylase with pyridoxal 5′-phosphate (PLP), 2′-methyl PLP (2′-MePLP), and pyridoxal 5′-deoxymethylenephosphonate (PDMP) results in enzymatically competent proteins. Pyridoxal in the absence and presence of phosphate and pyridoxal 5′-phosphate monomethyl ester are unable to form a Schiff base with apoOASS. The reconstitution of apoOASS with PLP is highly cooperative judged by the initial rate of activity regained and shows no evidence of saturation with PLP. The reconstituted enzymes have been studied using³¹P NMR spectroscopy. The³¹P NMR of the aposulfhydrylase reconstituted with PLP exhibits a chemical shift of 5.2 ppm, identical to that of native enzyme. The latter has been interpreted in terms of a strong ionic interaction between enzyme and the 5′-phosphate of PLP (P. F. Cook, S. Hara, S. Nalabolu, and K. D. Schnackerz, 1992,Biochemistry31, 2298–2303). Reconstitution with 2′-MePLP gives a lower chemical shift of 4.95 ppm, suggesting a weaker ionic interaction at the 5′-phosphate when compared to native enzyme. The PDMP-reconstituted enzyme gives a chemical shift of 23.7 ppm, consistent with the monoanionic form of the bound phosphonate. All of the chemical shifts are pH independent. The apoenzyme has also been reconstituted with pyri doxal 5′-sulfate. Although the resulting enzyme is not active in the overall reaction, it forms the external Schiff base. The PDMP- and 2′-MePLP-reconstituted enzymes have also been studied in the presence of amino acid reactants and analogues, and results are discussed in terms of the mechanism of OASS.
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1985, Biochemical and Biophysical Research Communications
The binding site of Pyridoxal-5-P in 4-aminobutyrate aminotransferase was studied by using analogs of the cofactor. A phosphorothioate analog (PLP(S)) recognizes the catalytic site; it forms a stable complex with the apoenzyme (K_D = 1nM) and serves as cofactor during catalysis. Replacement of a non-bridged oxygen by sulfur in the phosphate side chain renders a compound which preserves the negative charges needed for correct alignment of the cofactor at the catalytic site. This phosphorothioate analog of PLP can be used to investigate the catalytic site of vitamin B₆ dependent enzymes by means of ³¹P NMR spectroscopy.
A bulky P-pyridoxamine derivative, ie, N-4-azido-2-nitrophenyl pyridoxyl-5-P (NANP) competes with the natural cofactor for its binding site. Upon illumination, the arylazide of P-pyridoxamine acts as an efficient photolabeling reagent of the protein. A characteristic feature of this photolabeling reagent, ie, its ability to recognize the cofactor binding site, can be exploited to ascertain the chemical nature of amino acid residues at the catalytic site.
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2010, Comprehensive Natural Products II: Chemistry and Biology
Serine hydroxymethyltransferase: Role of Glu75 and evidence that serine is cleaved by a retroaldol mechanism
2004, Biochemistry

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Activation of aposerine transhydroxymethylase by pyridoxal-5′-phosphate monomethyl Ester

Summary

Vitamins and Hormones

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Am. Chem. Soc.