The near attack conformation approach to the study of the chorismate to prephenate reaction

Abstract

Standard free energies (⌬GN°) for formation of near attack conformers, those ground state conformers that can convert directly to the transition state, were calculated for the Claisen rearrangement of chorismate to prephenate in six different environments: water, wild-type enzymes from Bacillus subtilis and Escherichia coli, their Arg90Cit and Glu52Ala mutants, and the 1F7 catalytic antibody. Values of the calculated ⌬G N°s and the experimentally determined activation energies (⌬G ‡ ) are linearly related with the slope of Ϸ1. This demonstrates that the relative rate of the chorismate 3 prephenate reaction is overwhelmingly dependent on the efficiency of formation of near attack conformers in the ground state. In a chemical reaction of bond formation there is a time in which the two reacting atoms are at a distance of van der Waals contact and at an angle resembling the bond to be formed in the transition state (TS). By our convention this situation is called a near attack conformation (NAC) (1-3). The reaction of interest must proceed to the TS via NAC formation (Eq. 1). % of the population of ground state conformers in water, whereas in the enzyme, E⅐NAC consists of 30% of the Michaelis complex (3). It follows that ⌬G N°i n the water and the enzymatic reactions is 8.4 and 0.6 kcal͞mol, respectively, and accordingly, ⌬⌬G N°i s 7.8 kcal͞mol. Because the experimentally determined ⌬⌬G ‡ is 9 kcal͞mol, the advantage of the enzymatic reaction was concluded to be primarily in preferential formation of NACs. S L | ;We now report extensions of our studies using TI methods of free energies for NAC formation in water, Escherichia coli chorismate mutase and Bacillus subtilis chorismate mutase wildtype enzymes (w-EcCM and w-BsCM), the mutant obtained by alanine substitution for Glu-52 of EcCM (E52A), the mutant obtained by citrulline substitution for Arg-90 of BsCM (R90Cit), and the catalytic antibody 1F7 (4). MethodsFree energy calculations were performed by using TI methods (5) implemented in the program CHARMM V.27b4 (6). Free energy calculation for chorismate in water was performed on a dianionic chorismate solvated in a 24 ϫ 24 ϫ 24-Å 3 periodic box of TIP3P (7) water. The crystal structure of Protein Data Bank ID code 1ECM (8) was used for the w-EcCM and E52A simulations, the structure of ID code 2CHT (9) was used for the w-BsCM and R90Cit simulations, and the structure of ID code 1FIG (4) was used for the 1F7 simulations. The cocrystallized TS analogue [TSA (10); Scheme 1] was replaced by chorismate. The whole structure of each protein was solvated in a periodic box of TIP3P water and equilibrated at 300 K for 500 ps. The final MD structures were used to select residues within a 25-Å sphere from the active site. For the computational efficiency, only these selected residues were included for the TI calculations of protein systems with use of the stochastic boundary condition (6). The free energy derivatives were calculated every 5-10°of dih1 (dihedral angle of C4-C3-O13-C14; see Scheme 1). At each window, the system...

The proficiency of a thermophilic chorismate mutase enzyme is solely through an entropic advantage in the enzyme reaction

“…The free energy of activation in the enzymatic reaction is 9.93 kcal͞mol lower than the free energy of activation in water. The minimum of the free-energy profile of NAC formation in water [G NAC min ( Ј)] is 8.06 kcal͞mol, which is in good agreement with previous results (8.1 kcal͞mol) obtained from thermodynamic integration (4). In the enzymatic reaction, free energy for NAC formation is 0.73 kcal͞ mol when R C5-C16 is the distance of the NAC criteria (3.7 Å).…”

“…The free energies for NAC formation in the six different systems: water, WT EcCM and E52A mutant, WT BsCM and R90Citrulline mutant, and a catalytic antibody 1F7, were calculated by using thermodynamic integration (4). A linear plot (Fig.…”

“…Our choice to research the Thermus thermophilus chorismate mutase (TtCM) relates to our current interest in the chorismate 3 prephenate reaction (4)(5)(6) and the questions of the temperature specificity of thermophilic enzymes (7). TtCM belongs to a rare group of the AroH-type monofunctional CMs that are principally found in Gram-positive bacteria and have been discovered as a thermophilic enzyme in the Gram-negative T. thermophilus (8,9).…”

Conformational dependence of a protein kinase phosphate transfer reaction

“…This idea is similar in spirit to the near-attack conformation (NAC) of Bruice and coworkers (34) in that local geometry and stereo-chemistry considerations imply specific boundary conditions that will allow the reaction to proceed. The Red is oxygen, light blue is carbon, dark blue is nitrogen, silver is hydrogen, green is magnesium, and gold is phosphorus.…”

How an enzyme surmounts the activation energy barrier

“…The reaction itself is a vital step in the biosynthesis of aromatic amino acids, lending the enzyme a biological importance equal to its importance in understanding mechanisms of catalysis. In this issue of PNAS, Hur and Bruice (1) have applied the near-attack conformation (NAC) concept, which Bruice and coworkers have been developing for several years (2,3), to the accelerations produced by several different chorismate mutases, developing thereby what might be called a microhistory of the catalytic process for these enzymes, tracing events between reactant state and transition state. The findings are illuminating and likely to generate considerable surprise.…”