Spinach chloroplast 0-acetylserine (thiol)-lyase exhibits two catalytically non-equivalent pyridoxal-5'-phosphate-containing active sites.
A synthetic gene encoding the mature spinach- chloroplast O-acetylserine (thiol)-lyase was constructed and expressed in an Escherichia coli strain carrying the T7 RNA polymerase system. The pure recombinant protein was obtained at high yield (6 mg/l cell culture) using a new purification procedure that includes affinity chromatography on Green A agarose. Its specific activity was of the order of 1000 U/mg, and its physical properties were similar to those previously reported for the natural enzyme isolated from spinach chloroplasts. In particular the recombinant enzyme, as for the natural enzyme, behaved as a homodimer composed of two identical subunits each of Mr 35000. From steady-state kinetic studies using sulfide or 5-thio(2-nitrobenzoate) (Nbs) as alternative nucleophilic co-substrates, the enzyme exhibited positive kinetic co-operativity with respect to O-acetylserine [Ser(Ac)] in the presence of sulfide and a negative kinetic co-operativity in the presence of Nbs. Binding of Ser(Ac) to the enzyme was also investigated by absorbance and fluorescence measurements to obtain insight into the role of pyridoxal 5'-phosphate and of the single tryptophan residue (Trp176) present in the enzyme molecule. Addition of Ser(Ac) to the enzyme provoked the disappearance of the 409-nm absorbance band of the pyridoxal 5'-phosphate Schiff base and the appearance of two new absorbance bands, the one located between 320 nm and 360 nm and the other centered at 470 nm. Also, the fluorescence emission of the pyridoxal 5'-phosphate Schiff base was quenched upon addition of Ser(Ac) to the enzyme. These changes were most presumably due to the formation of a Schiff base intermediate between alpha-aminoacrylate and the pyridoxal 5'-phosphate cofactor. The fluorescence emission of Trp176 was also quenched upon Ser(Ac) binding to the enzyme. Quantitative analysis of the absorbance and fluorescence equilibrium data disclosed a co-operative behavior in Ser(Ac) binding, in agreement with the steady-state kinetic results. Fluorescence quenching experiments with the acrylamide and iodide revealed that the indole ring of Trp176 was largely exposed and located within the pyridoxal 5'-phosphate active site. These results are consistent with the finding that the native enzyme is composed of two identical subunits. Yet, presumably due to subunit-subunit interactions, the enzyme exhibits two non-equivalent pyridoxal-5'-phosphate-containing active sites.
Rolland
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Unité Mixte C.N.R.S./Rhône-Poulenc, U.M. 41, Lyon, France.
Ruffet
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ML
Job
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D
Douce
R
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Droux
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D003547:Cysteine Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D005470:Fluorometry; D017343:Genes, Plant; D005813:Genes, Synthetic; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D018724:Spinacia oleracea / Q000201:enzymology / Q000235:genetics; D017186:Titrimetry; D014364:Tryptophan / Q000737:chemistry
D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D011732:Pyridoxal Phosphate; D014364:Tryptophan; D003547:Cysteine Synthase; C103381:MET17 protein, S cerevisiae; C018725:O-acetylhomoserine (thiol)-lyase; D008190:Lyases; D019757:Carbon-Oxygen Lyases