Catalytic residues and an electrostatic sandwich that promote enolpyruvyl shikimate 3-phosphate synthase (AroA) catalysis.
Enolpyruvylshikimate 3-phosphate synthase (EPSP synthase, AroA) catalyzes the sixth step in aromatic amino acid biosynthesis. It forms EPSP from shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) in an addition/elimination reaction that proceeds through a tetrahedral intermediate. In spite of numerous mechanistic studies, the catalytic roles of specific amino acid residues remain an open question. Recent experimental evidence for cationic intermediates or cationic transition states, and a consideration of the catalytic imperative, have guided this study on the catalytic roles of Lys22 (K22), Asp313 (D313), and Glu341 (E341). Steady-state and pre-steady-state kinetics and protein stability studies showed that mutations of D313 and E341 caused k(cat) to decrease up to 30,000-fold and 76,000-fold, respectively, while the effects on K(M) were modest, never more than 40-fold. Thus, both are identified as catalytic residues. In an active site that is overwhelmingly positively charged, the D313 and E341 side chains are positioned to form an "electrostatic sandwich" around the positive charge at C2 in cationic intermediates/transition states, stabilizing them and thereby promoting catalysis. Mutation of K22 showed large effects on K(M,S3P) (100-fold), K(M,PEP) (>760-fold), and up to 120-fold on k(cat). Thus, K22 had roles in both substrate-binding and transition-state stabilization. These results support the identification of E341 and K22 as general acid/base catalytic residues.
Berti
Paul J
PJ
Department of Chemistry, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4M1, Canada. berti@mcmaster.ca
Chindemi
Paul
P
D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001224:Aspartic Acid / Q000096:biosynthesis / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology*; D002412:Cations / Q000737:chemistry; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D005971:Glutamates / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008239:Lysine / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013379:Substrate Specificity / Q000235:genetics
D002412:Cations; D029968:Escherichia coli Proteins; D005971:Glutamates; D012765:Shikimic Acid; D001224:Aspartic Acid; C042786:shikimic acid-3-phosphate; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D008239:Lysine