The formation of a complex between p21(ras) and GAP accelerates the GTPase reaction of p21(ras) and terminates the signal for cell proliferation. The understanding of this rate acceleration is important for the elucidation of the role of Ras mutants in tumor formation. In principle there are two main options for the origin of the effect of GAP. One is a direct electrostatic interaction between the residues of GAP and the transition state of the Ras-GAP complex and the other is a GAP-induced shift of the structure of Ras to a configuration that increases the stabilization of the transition state. This work examines the relative importance of these options by computer simulations of the catalytic effect of Ras. The simulations use the empirical valence bond (EVB) method to study the GTPase reaction along the alternative associative and dissociative paths. This approach reproduces the trend in the overall experimentally observed catalytic effect of GAP: the calculated effect is 7 +/- 3 kcal/mol as compared to the observed effect of approximately 6.6 kcal/mol. Furthermore, the calculated effect of mutating Arg789 to a nonpolar residue is 3-4 kcal/mol as compared to the observed effect of 4.5 kcal/mol for the Arg789Ala mutation. It is concluded, in agreement with previous proposals, that the effect of Arg789 is associated with its direct interaction with the transition state charge distribution. However, calculations that use the coordinates of Ras from the Ras-GAP complex (referred to here as Ras') reproduce a significant catalytic effect relative to the Ras coordinates. This indicates that part of the effect of GAP involves a stabilization of a catalytic configuration of Ras. This configuration increases the positive electrostatic potential on the beta-phosphate (relative to the corresponding situation in the free Ras). In other words, GAP stabilizes the GDP bound configuration of Ras relative to that of the GTP-bound conformation. The elusive oncogenic effect of mutating Gln61 is also explored. The calculated effect of such mutations in the Ras-GAP complex are found to be small, while the observed effect is very large (8.7 kcal/mol). Since the Ras is locked in its Ras-GAP configuration in our simulations, we conclude that the oncogenic effect of mutation of Gln61 is indirect and is associated most probably with the structural changes of Ras upon forming the Ras-GAP complex. In view of these and the results for the Ras' we conclude that GAP activates Ras by both direct electrostatic stabilization of the transition state and an indirect allosteric effect that stabilizes the GDP-bound form. The present study also explored the feasibility of the associative and dissociative mechanism in the GTPase reaction of Ras. It is concluded that the reaction is most likely to involve an associative mechanism.

The idea that enzymes accelerate their reactions by entropic effects has played a major role in many prominent proposals about the origin of enzyme catalysis. This idea implies that the binding to an enzyme active site freezes the motion of the reacting fragments and eliminates their entropic contributions, (⌬S cat ‡ )Ј, to the activation energy. It is also implied that the binding entropy is equal to the activation entropy, (⌬S w ‡ )Ј, of the corresponding solution reaction. It is, however, difficult to examine this idea by experimental approaches. The present paper defines the entropic proposal in a rigorous way and develops a computer simulation approach that determines (⌬S ‡ )Ј. This approach allows us to evaluate the differences between (⌬S ‡ )Ј of an enzymatic reaction and of the corresponding reference reaction in solution. Our approach is used in a study of the entropic contribution to the catalytic reaction of subtilisin. It is found that this contribution is much smaller than previously thought. This result is due to the following: (i ) Many of the motions that are free in the reactants state of the reference solution reaction are also free at the transition state. Many prominent proposals (e.g., see refs. 1 and 2) and textbooks that consider biochemical systems (e.g., refs. 3 and 4) invoke entropic contributions as major factors in enzyme catalysis. These proposals, which are intuitively very appealing (e.g., see ref. 5), have assumed that the large configurational space available for the reacting fragments in water would be drastically restricted in the enzyme active site. It has been thus deduced that this should lead to large entropic contributions to the difference between the activation barrier in the enzyme and in the reference solution reaction. However, the validity of these proposals is far from being obvious (6, 7). For example, the very inf luential proposal introduced by Page and Jencks (1) ref lects the assumption that the formation of the transition state in a bimolecular reaction in solution involves complete loss of three translational and three rotational degrees of freedom. However, two or more of these degrees of freedom are usually almost free in the transition state (see below). More serious is the implicit assumption that the entropic contribution to catalysis is given approximately by the negative of the binding entropy (see below). Other problems with simple estimates of the entropic contribution will be mentioned in the next section.The main stumbling block for determining the validity of the entropic proposal is the absence of direct experimental information about the corresponding contribution of the reacting fragments to the activation entropy in the enzyme and in solution. In this respect it is interesting to note the recent analysis of cytidine deaminase by Wolfenden and co-workers (8). This study found that the entropies of activation in the enzyme and in water are very similar and that the overall catalysis is due to enthalpic effects. Interestingly, it was found that ...

A force field for monosaccharides that can be extended to (1 + 4) linked polysaccharides has been developed for the AMBER potential function. The resulting force field is consistent with the existing AMBER force field for proteins and nucleic acids. Modifications to the standard AMBER OH force constant and to the Lennard-Jones parameters were made. Furthermore, a 10-12 nonbonded term was included between the hydroxyl hydrogen of the saccharide and the water oxygen (TIP3P, SPC/E, etc.) to reproduce better the water-saccharide intermolecular distances. STO-3G electrostatic potential (ESP) charges were used to represent the electrostatic interactions between the saccharide and its surrounding environment. To obtain charges for polysaccharides, a scheme was developed to piece together saccharide residues through 1 + 4 connections while still retaining a net neutral charge on the molecule as a whole. Free energy perturbation (FEP) simulations of Dglucose and D-mannose in water were performed to test the resulting force field. The FEP simulations demonstrate that AMBER overestimates intramolecular interaction energies, suggesting that further improvements are needed in this part of the force field. To test further the reliability of the parameters, a molecular dynamics (MD) simulation of a-D-glucose in water was also performed. The MD simulation was able to produce structural and conformational results that are in accord with experimental evidence and previous theoretical results. Finally, a relaxed conformational map of pmaltose was assembled and it was found that the present force field is consistent with available theoretical and experimental results. 0 1994 by John Wiley & Sons, InC.

The energetics of the catalytic reaction of ribonuclease A have been explored with empirical valence bond (EVB) simulations to elucidate the origin of the enormous catalytic power of this enzyme and to examine different mechanistic alternatives. The two mechanisms analyzed were a general acid-general base mechanism with a dianionic transition state and a triester-like mechanism with a monoionic transition state. The first step of the analysis used experimental information to determine the activation energy of each assumed mechanism in a water cage. This has provided an experimentally based reference point for the catalytic effect of the enzyme. The next step of the analysis involved EVB simulations of the reaction in water and calibrated these simulations against the above-mentioned energetics of the reference reaction. The simulations were performed in the protein environment without changing any EVB parameters. The catalysis was measured as the difference in the overall activation free energy of the respective mechanism in water and in protein. In the mechanism with the dianionic transition state a catalytic effect of ∼18 ( 6 kcal/mol was established which is in good agreement with the experimentally derived estimate of ∼21 kcal/mol. In the mechanism with the monoionic transition state, a value of ∼12 ( 6 kcal/mol was calculated which is 5 kcal/mol lower than its estimated value, making it slightly less likely to be the actual mechanism in the enzyme. The origin of the catalytic power is attributed to an electrostatic reduction of the activation barriers. This reduction is associated with the preorganized polar environment of the enzyme.

Quantum mechanics calculations and molecular mechanics calculations using the MMX force field have been performed on the low-energy conformations of 9-crown-3. A [144] conformation corresponds to the global minimum at the MP2/6-31G**//6-31G** level, with a [225] conformation only 0.72 kcal/mol higher in energy. The [333] conformation in which all three oxygen atoms are on the same side of the least squares plane passing through the ring, a structure particularly favorable as a tridentate ligand, is calculated to be 6.81 kcal/mol higher in energy. Electron correlation at the MP2 level and polarization functions on the hydrogen atoms have only minor effects on the relative conformational energies. However, polarization functions on the carbon and oxygen atoms are needed to obtain good results. MMX conformational energies are in good agreement with ab initio values if the electrostatic scale factor is set at 0.70. The role of transannular C-H‚‚‚O hydrogen bonding in the low-energy conformations of 9-crown-3 is discussed. Results are compared with those obtained from AM1, PM3, and AMBER calculations.