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Binding TS in preference to S and increasing TDeltaS++by freezing out motions in E X S and E X TS have been accepted as the driving forces in enzymatic catalysis; however, the smaller value of DeltaG++ for a one-substrate enzymatic reaction, as compared to its nonenzymatic counterpart, is generally the result of a smaller value of DeltaH++. Ground-state conformers (E X NACs) are formed in enzymatic reactions that structurally resemble E X TS. E X NACs are in thermal equilibrium with all other E X S conformers and are turnstiles through which substrate molecules must pass to arrive at the lowest-energy TS. TS in E X TS may or may not be bound tighter than NAC in E X NAC.

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...

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