Identification of the catalytic residues of AroA (Enolpyruvylshikimate 3-phosphate synthase) using partitioning analysis.
AroA (EPSP synthase) catalyzes carboxyvinyl transfer through addition of shikimate 3-phosphate (S3P) to phosphoenolpyruvate (PEP) to form a tetrahedral intermediate (THI), followed by phosphate elimination to give enolpyruvylshikimate 3-phosphate (EPSP). A novel approach, partitioning analysis, was used to elucidate the roles of catalytic residues in each step of the reaction. Partitioning analysis involved trapping and purifying [1-(14)C]THI, degrading it with AroA, and quantitating the products. Wild-type AroA gave a partitioning factor, f(PEP) = 0.25 +/- 0.02 at pH 7.5, where f(PEP) = [[1-(14)C]PEP]/([[1-(14)C]PEP] + [[1-(14)C]EPSP]). Eighteen mutations were made to 14 amino acids to discover which residues preferentially catalyzed either the addition or the elimination step. Mutating a residue catalyzing one step (e.g., addition) should change f(PEP) to favor the opposite step (e.g., elimination). No mutants caused large changes in f(PEP), with experimental values from 0.07 to 0.41. This implied that there are no side chains that catalyze only addition or elimination, which further implied that the same residues are general acid/base catalysts in both forward and reverse THI breakdown. Only Lys22 (protonating S3P hydroxyl or phosphate) and Glu341 (deprotonating C3 of PEP) are correctly situated in the active site. In the overall reaction, Lys22 would act as a general base during addition, while Glu341 would act as a general acid. Almost half of the mutations (eight of 18) caused a >1000-fold decrease in specific activity, demonstrating that a large number of residues are important for transition state stabilization, "ensemble catalysis", in contrast to some enzymes where a single amino acid can be responsible for up to 10(8)-fold catalytic enhancement.
Mizyed
Shehadeh
S
Department of Chemistry, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4M1, Canada.
Wright
Jennifer E I
JE
Byczynski
Bartosz
B
Berti
Paul J
PJ
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites; D002247:Carbon Isotopes; D002384:Catalysis; D003198:Computer Simulation; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009716:Numerical Analysis, Computer-Assisted; D010728:Phosphoenolpyruvate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D002247:Carbon Isotopes; D011994:Recombinant Proteins; D012765:Shikimic Acid; D018698:Glutamic Acid; D010728:Phosphoenolpyruvate; D019883:Alkyl and Aryl Transferases; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D008239:Lysine