Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plants--structural and kinetic properties of the free and bound enzymes. Export

@article{citeulike:3874606, abstract = {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.}, author = {Droux, M. and Ruffet, M. L. and Douce, R. and Job, D.}, citeulike-article-id = {3874606}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/9692924}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=9692924}, day = {1}, issn = {0014-2956}, journal = {European journal of biochemistry / FEBS}, keywords = {acetyltransferase, channelingexample, plants, serine}, month = {July}, number = {1}, pages = {235--245}, posted-at = {2009-01-11 20:31:41}, priority = {2}, title = {Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plants--structural and kinetic properties of the free and bound enzymes.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/9692924}, volume = {255}, year = {1998} }

TY - JOUR ID - citeulike:3874606 L3 - citeulike-article-id:3874606 N2 - 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. IS - 1 JF - European journal of biochemistry / FEBS SN - 0014-2956 EP - 245 TI - Interactions between serine acetyltransferase and O-acetylserine (thiol) lyase in higher plants--structural and kinetic properties of the free and bound enzymes. VL - 255 SP - 235 KW - acetyltransferase KW - channelingexample KW - plants KW - serine AU - Droux, M AU - Ruffet, ML AU - Douce, R AU - Job, D PY - 1998/07/01/ UR - http://view.ncbi.nlm.nih.gov/pubmed/9692924 ER -