The detailed mechanism of DNA hydrolysis by enzymes is of significant current interest. One of the most important questions in this respect is the catalytic role of metal ions such as Mg2+. While it is clear that divalent ions play a major role in DNA hydrolysis, it is uncertain what function such cations have in hydrolysis and why two are needed in some cases and only one in others. Experimental evaluation of the catalytic effects of the cations is problematic, since the cations are intimately involved in substrate binding. This problem is explored here by using a theoretical approach to analyze and interpret the key structural and biochemical experiments. Taking the X-ray structure of the exonuclease domain in the Klenow fragment of E. coli DNA polymerase I we use the empirical valence bond method to examine different feasible mechanisms for phosphodiester bond cleavage in the exonuclease site. This structure-function analysis is based on evaluating the activation free energies of different assumed mechanisms and comparing the calculated values to the corresponding experimentally observed activation energy for phosphodiester bond cleavage. Mechanisms whose calculated activation energies are drastically larger than the observed activation energy are eliminated and the consistency of the corresponding conclusion is examined in view of other available experimental facts including mutational and pH dependence studies. This approach indicates that phosphodiester bond hydrolysis involves catalysis by an OHion from aqueous solution around the protein, rather than a general base catalysis by an active site residue. The catalytic effect of two divalent metal cations in the active site is found to be primarily electrostatic. The first cation provides a strong electrostatic stabilization to the OHñ ucleophile, while the second cation provides a very large catalytic effect by its interaction with the negative charge being transferred to the transition state during the nucleophilic attack step. The calculations also demonstrate that the second metal ion is not likely to be involved in a previously proposed strain mechanism. The two-metal ion catalytic mechanism is compared to the action of a single-metal cation active site and some general rules are discussed.Finally the relationship between the present computer modeling study and available experimental information on DNA hydrolysis is discussed, emphasizing that calculations of absolute rate constants should be, at least in principle, more effective in eliminating incorrect mechanisms than calculations of mutational effects. 1. Introduction Recent structural and biochemical studies have shed new light on the molecular details of both the hydrolysis and

A major challenge for theoretical simulation methods is the calculation of enzymic reaction rates directly from the three-dimensional protein structure together with some idea of the chemical reaction mechanism. Here, we report the evaluation of a complete free energy profile for all the elementary steps of the triosephosphate isomerase catalyzed reaction using such an approach. The results are compatible with available experimental data and also suggest which of the possible reaction intermediates is kinetically observable. In addition to previously identified catalytic residues, the simulations show that a crystallographically observed active site water molecule plays an important role during catalysis and an intersubunit interaction that could explain the low activity of the monomeric enzyme is also observed. The calculations clearly demonstrate the important catalytic effects associated with stabilization of charged high energy intermediates and reduction of reorganization energy, which are likely to be general principles of enzyme catalyzed charge transfer and separation reactions.

Recent studies of genetically modified enzymes have indicated that changes in activation free energies, AAg*, and changes in reaction free energies, AAGq, are correlated by the relationship AAg* = ß • The present work explores the basis for such linear free energy relationships (LFERs) in enzymatic reactions, focusing on the effects of mutations in tyrosyl-tRNA-synthetase (TTS). It is demonstrated that the optimal way to analyze LFERs is by describing the reaction in terms of pure valence bond (VB) resonance structures rather than in terms of partially formed bonds. The use of the pure VB representation allows one to evaluate the relevant LFER using Marcus-type concepts and to compare the predicted ß to the observed one. Using a two-resonance-structure VB model for TTS produces ß 0.5, which disagrees with the observed values of ß ^0.83 and ß » 1 for two classes of mutations. Noting, however, that the phosphoryl transfer process in TTS has been described before as going through a high-energy intermediate, we describe this reaction in terms of three VB resonance structures. This accounts for the observed values of ß and supports the validity of LFER in TTS. It is pointed out that LFERs are valid in proteins even when the changes in Ag* involve very anharmonic interactions like hydrogen bonds, since such relationships reflect the correlation between AAg* and AAGq rather than the correlation between AAg* and the effect of specific residues. However, obtaining LFER in proteins requires that the active site environment responds linearly to the change of charges during the reaction, and such a linear response is far from obvious. Fortunately, the simulation study presented in this work as well as previous simulations has demonstrated that active sites of proteins obey the linear response approximation. Such a behavior of highly anharmonic systems is due to the availability of many compensating polar interactions. This finding provides a theoretical basis for the experimental observation of LFER in TTS.

larger alkyl groups, and other substituents, X. It could be extended to di-, tri-, and polysubstituted alkanes if induced dipoles or van der Waals forces are includedLLJ5 Following Benson and Luna4 it could be extended to radicals and alkenes. Once it has been refined, it could be made accessible in molecular mechanics programs, where it might reduce the number of input parameters required or provide better understanding of the forces involved.With the inclusion of appropriate induced dipoles, it could be used to calculate molecular dipole moments and intermolecular forces.As it stands, the method does provide a simple, quantitative explanation for the interesting effects of methyl groups on molecular stability in Table 11; an explanation which has been lacking for more than 20 years.' Acknowledgment. The authors wish to thank Y.-R. Luo, T.

The first step in the assimilation of atmospheric CO 2 into biomass, carboxylation of D-ribulose 1,5-bisphosphate (RuBP)