Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plants--structural and kinetic properties of the free and bound enzymes.
The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L-serine in the presence of acetyl-CoA to form O-acetylserine. The second enzyme, O-acetylserine (thiol) lyase, converts O-acetylserine to L-cysteine in the presence of sulfide. We have, in the present work, over-produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O-acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high-molecular-mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O-acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O-acetylserine (thiol) lyase to form a bienzyme complex (molecular mass approximately 300 kDa) called cysteine synthase containing 4 mol pyridoxal 5'-phosphate/mol complex. O-Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O-acetylserine. Protein-protein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase: there was a transition from a typical Michaelis-Menten model to a model displaying positive kinetic co-operativity with respect to serine and acetyl-CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O-acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build-up of O-acetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the presence of a large excess of free O-acetylserine (thiol) lyase. These findings contradict the widely held belief that such a bienzyme complex is required to channel the metabolite intermediate O-acetylserine.
Droux
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Laboratoire Mixte CNRS/Rhône-Poulenc (UMR041), Rhône-Poulenc Agrochimie, Lyon, France. michel.droux@ladargoire.rhone-poulenc.com
Ruffet
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Douce
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D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology*; D002736:Chloroplasts / Q000201:enzymology; D003545:Cysteine / Q000096:biosynthesis*; D003547:Cysteine Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism; D051050:Serine O-Acetyltransferase; D013045:Species Specificity; D017186:Titrimetry
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D012694:Serine; D000123:Acetyltransferases; D051050:Serine O-Acetyltransferase; D003547:Cysteine Synthase; C043943:O-acetylserine; D003545:Cysteine