title: Yeast chorismate mutase in the R state: simulations of the active site.
abstract: The isomerization of chorismate to prephenate by chorismate mutase in the biosynthetic pathway that forms Tyr and Phe involves C5---O (ether) bond cleavage and C1---C9 bond formation in a Claisen rearrangement. Development of negative charge on the ether oxygen, stabilized by Lys-168 and Glu-246, is inferred from the structure of a complex with a transition state analogue (TSA) and from the pH-rate profile of the enzyme and the E246Q mutant. These studies imply a protonated Glu-246 well above pH 7. Here, several 500-ps molecular dynamics simulations test the stability of enzyme-TSA complexes by using a solvated system with stochastic boundary conditions. The simulated systems are (i) protonated Glu-246 (stable), (ii) deprotonated Glu-246 (unstable), (iii) deprotonated Glu-246 plus one H2O between Glu-246 and the ether oxygen (unstable), (iv) the E246Q mutant (stable), and (v) addition of OH- between protonated Glu-246 and the ether oxygen. In (v), a local conformational change of Lys-168 displaced the OH- into the solvent region, suggesting a possible rate-determining step that precedes the catalytic step. In a 500-ps simulation of the enzyme complexed with the reactant chorismate or the product prephenate, no water molecule remained near the oxygen of the ligand. Calculations using the linearized Poisson-Boltzmann equation show that the effective pKa of Glu-246 is shifted from 5.8 to 8.1 as the negative charge on the ether oxygen of the TSA is changed from -0.56 electron to -0.9 electron. Altogether, these results support retention of a proton on Glu-246 to high pH and the absence of a water molecule in the catalytic steps.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
authors.forename: J
authors.initials: J
authors.lastname: Ma
authors.forename: X
authors.initials: X
authors.lastname: Zheng
authors.forename: G
authors.initials: G
authors.lastname: Schnappauf
authors.forename: G
authors.initials: G
authors.lastname: Braus
authors.forename: M
authors.initials: M
authors.lastname: Karplus
authors.forename: W N
authors.initials: WN
authors.lastname: Lipscomb
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D002826:Chorismate Mutase / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae
chemical_list: D007527:Isoenzymes; D002826:Chorismate Mutase