We present an atomic-level description of the reaction chemistry of an enzyme-catalyzed reaction dominated by proton tunneling. By solving structures of reaction intermediates at near-atomic resolution, we have identified the reaction pathway for tryptamine oxidation by aromatic amine dehydrogenase. Combining experiment and computer simulation, we show proton transfer occurs predominantly to oxygen O2 of Asp(128)beta in a reaction dominated by tunneling over approximately 0.6 angstroms. The role of long-range coupled motions in promoting tunneling is controversial. We show that, in this enzyme system, tunneling is promoted by a short-range motion modulating proton-acceptor distance and no long-range coupled motion is required.

The accurate prediction of enzyme kinetics from first principles is one of the central goals of theoretical biochemistry. Currently there is considerable debate [1][2][3] about the applicability of transition state theory (TST) to compute rate constants of enzyme-catalyzed reactions. Classical TST is known to be insufficient in some cases, but corrections for dynamical recrossing and quantum mechanical tunneling can be included. [1,2,4] Many effects that go beyond the framework of TST have been proposed, particularly focusing on the possible role of protein dynamics and conformational effects on the enzyme activity. Unfortunately, the overall importance of these effects for the effective reaction rate is difficult (if not impossible) to determine experimentally. However, if one could compute the quasi-thermodynamical free energy of activation with chemical accuracy (i.e. 1 kcal mol À1 ), comparison with the effective measured free energy of activation would directly show the importance of other effects.Combined quantum mechanical/molecular mechanical (QM/MM) methods have become an important tool for computational modeling of enzyme-catalyzed reactions. Only the substrate(s) and relevant residues in the active site are treated quantum mechanically, the rest of the protein is described at the empirical MM level. This lowers the computational expense, making it possible to treat large enzyme systems and the surrounding solvent, and to sample phase space. Nevertheless, the number of QM atoms is still relatively large, and until now only low levels of QM theory, such as semiempirical methods or density functional theory (DFT), have been feasible. Semiempirical methods, though applicable to large systems, are generally not accurate enough because computed free energies of activation may have an error of ten or more kcal mol À1 . DFT (especially with the B3LYP functional [5] ) offers improved accuracy but still lacks key physical interactions (e.g., dispersion). Often, DFT underestimates barrier heights by several kcal mol À1 , which cannot be systematically improved. Thus, when theoretical barriers do not agree with those from experiment, it is not clear whether the discrepancy arises from deficiencies in the electronic structure theory and the sampling, in the experimental observations, or in the underlying theoretical framework of QM/MM and TST.Consequently, there is a need for high-level electronic structure calculations for reliable predictions of enzyme reactivity. The ab initio electron correlation methods MP2 (Møller-Plesset second-order perturbation theory), CCSD (coupled-cluster theory with single and double excitations), and CCSD(T) (CCSD with a perturbative treatment of triple excitations) provide a well-established hierarchy that converge reliably to give high accuracy, and rate constants of gasphase reactions involving only a few atoms can be predicted with error bars comparable to those found experimentally. [6,7] However, the computational expense of these methods increases very rapidly with the number o...

Chorismate mutase is a key model system in the development of theories of enzyme catalysis. To analyze the physical nature of catalytic interactions within the enzyme active site and to estimate the stabilization of the transition state (TS) relative to the substrate (differential transition state stabilization, DTSS), we have carried out nonempirical variation-perturbation analysis of the electrostatic, exchange, delocalization, and correlation interactions of the enzyme-bound substrate and transition-state structures derived from ab initio QM/MM modeling of Bacillus subtilis chorismate mutase. Significant TS stabilization by approximately -23 kcal/mol [MP2/6-31G(d)] relative to the bound substrate is in agreement with that of previous QM/MM modeling and contrasts with suggestions that catalysis by this enzyme arises purely from conformational selection effects. The most important contributions to DTSS come from the residues, Arg90, Arg7, Glu78, a crystallographic water molecule, Arg116, and Arg63, and are dominated by electrostatic effects. Analysis of the differential electrostatic potential of the TS and substrate allows calculation of the catalytic field, predicting the optimal location of charged groups to achieve maximal DTSS. Comparison with the active site of the enzyme from those of several species shows that the positions of charged active site residues correspond closely to the optimal catalytic field, showing that the enzyme has evolved specifically to stabilize the TS relative to the substrate.