Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. The catalytic rate in this system has been found to be significantly affected by mutations far from the site of chemical activity in the enzyme [Rajagopalan, P. T. R, Lutz, S., and Benkovic, S. J. (2002) Biochemistry 41, 12618 -12628]. On the basis of extensive computer simulations for wild-type DHFR from Escherichia coli and four mutants (G121S, G121V, M42F, and M42F͞ G121S), we show that key parameters for catalysis are changed. The parameters we study are relative populations of different conformations sampled and hydrogen bonds. We find that the mutations result in long-range structural perturbations, rationalizing the effects that the mutations have on the kinetics of the enzyme. Such perturbations also provide a rationalization for the reported nonadditivity effect for double mutations. We finally examine the role a structural perturbation will have on the hydride transfer step. On the basis of our new findings, we discuss the role of coupled motions between distant regions in the enzyme, which previously was reported by Radkiewicz and Brooks.

Aspartic acid and asparagine residues racemize rapidly relative to other amino acid residues in proteins and peptides. This has been attributed to the increased acidity of the α-carbons of succinimide residues derived from the spontaneous cyclizations of these residues. To understand the basis of this effect, the acidities of model compounds were calculated using ab initio quantum mechanics (RHF/6-31+G*). The results were also checked with DFT (Becke3LYP/6-31+G*) and solvent cavity models (IPCM and SCIPCM). The geometries of succinimide, 2-pyrrolidinone, and the derived enolate anions were optimized, and the gas phase deprotonation energies were calculated. The imide is more acidic than the amide by 18 kcal/mol in the gas phase. Since there is a qualitative correlation between gas phase and aqueous acidities, this result provides an explanation for the experimental observations that the rate of peptidyl succinimide racemization can be ∼105 times greater than that of unmodified aspartic acid residues. To quantitate the source of the succinimide acidity, the geometries and CH acidities of various conformations of N-formylacetamide and acetamide, acyclic models of succinimide and 2-pyrrolidinone, and 3-oxobutanal and acetone, acyclic models lacking the nitrogen atom, were studied. The importance of resonance effects for increasing the acidity of the α-carbon of succinimide was established, but electrostatic and inductive effects also have an important influence on acidities. The acidity of succinimide is compared to the acidities of several peptide models. Isosuccinimide, an alternative degradation product of aspartic acid and asparagine residues, is also be expected to be racemization prone by similar mechanisms.

The rate of spontaneous degradations of asparagine and aspartyl residues occurring through succinimide intermediates is dependent upon the nature of the residue on the carboxyl side in peptides. For nonglycine residues, we show here that this effect can largely be attributed to the electrostatic/inductive effect of the side chain group on the equilibrium concentration of the anionic form of the peptide bond nitrogen atom that initiates the succinimide forming reaction. However, the rate of degradation of Asn-Gly and Asp-Gly containing peptides is about an order of magnitude greater than predicted solely using this explanation. To understand the nature of the glycine effect, ab initio calculations were performed on model compounds. These calculations indicate that there is little to no change in the stability of the transition state or the tetrahedral intermediate of succinimide formation with Asn-/Asp-Gly and Asn-/Asp-Ala derivatives. However, we have found that the acidity of the backbone peptide nitrogen NH is highly dependent upon the conformation of the molecule. Since glycine residues lack the beta-carbon common to all other protein amino acids, these residues can sample additional regions of conformational space where it is possible to further stabilize the backbone amide anion and thus increase the rate of degradation. These results provide the first rationale for the particular rate enhancement of degradation in peptidyl Asn-/Asp-Gly sequences. The results also can be applied to asparagine and aspartyl residues in proteins where the 3-dimensional structure provides additional constraints on conformation that can either increase or decrease the equilibrium concentration of the backbone amide anion and thus their rate of degradation via succinimide intermediates. Understanding this chemistry will assist attempts to minimize the deleterious effect of aging at the molecular level. The relationship between these results and proton exchange experiments is discussed in the Appendix.

The conformations and electrostatic potentials of phosphonamides, phosphonamidates and sulfonamides have been compared to the tetrahedral intermediate for base-catalyzed amide hydrolysis. The wide variation in inhibition by these similar compounds is explained through differences in electrostatic effects.