Identification of the Catalytic Residues of AroA (Enolpyruvylshikimate 3-Phosphate Synthase) Using Partitioning Analysis

Abstract

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.

Phosphate Analogues as Probes of the Catalytic Mechanisms of MurA and AroA, Two Carboxyvinyl Transferases

“…Its kcatfKm was 4 x 10 3 M-1 s-, 3 x 1 0 4 -fold slower than AreA-catalyzed THI breakdown (30). Presumably this was due to slower binding.…”

“…In AroA, Glu341, the equivalent residue to Cys115 in MurA, was proposed to act dual role in both protonation of C3 of PEP and deprotonation of THI (30).…”

“…Now the question is how the enzyme evolved itself to discriminate one single proton. An unexpected result from AroA mutation studies was a single mutation 049A decreased AroAH 6 activity by 21 000-fold (30). Based on crystal structures, except for the presumed catalytic base E341 in AroA and C115 in MurA, which was used to deprotonate C3 hydrogen, 049 is the only negatively charged residue around phosphate.…”

“…Based on crystal structures, except for the presumed catalytic base E341 in AroA and C115 in MurA, which was used to deprotonate C3 hydrogen, 049 is the only negatively charged residue around phosphate. This residue is 100% conserved in both AroA and MurA (30). The enzymes use several hydrogen bond donor resides (K22, R100, R124, 0171 in AroA and K22, R91, R120 in MurA) to stabilize negative charge on phosphate oxygens.…”

UDP-N-acetilglicosamina enolpiruvil transferase: determinação dos estados de protonação de resíduos de aminoácidos do sítio ativo pelo método PM6

“…Os mecanismos das reações catalisadas pela MurA e AroA ainda não são completamente conhecidos, porém há propostas para esses mecanismos que foram construídas a partir de observações experimentais. [6][7][8][9] Estudos sugerem que essas reações ocorrem em duas etapas, sendo a primeira de adição e a segunda de eliminação, com a formação de um intermediário tetraédrico (IT) entre elas (Esquemas 2a e 2b). Entretanto, ainda não foi esclarecido de forma inequívoca quais são os aminoácidos que atuam como ácido e base em cada etapa.…”