Thomas C. Bruice

Normal mode analysis, using the elastic network model, has been employed to envision the low frequency normal mode motion trends in the structures of five intermediates and a transition state in the kinetic pathway of E. coli dihydrofolate reductase (DHFR). Five of the reaction pathway analog structures and a crystal structure resembling the transition state, using X-ray analyses determined by Kraut et al., have been adapted as structural models. The motions that poise pathways of the M20 loop transitions from closed to occluded conformations and sub domain rotation to close the substrate cleft, have been predicted and envisioned for the first time by this study. Pathway entries to the movement of the substrate binding cleft helices are also envisioned. These motions play roles in transition structure stabilization and in regulating the release of the product tetrahydrofolate (THF). The motions observed push the ground state conformation of each intermediate towards a higher energy sub state conformation. A set of conserved residues involved in the catalytic reactions and conformational changes, previously studied by kinetic, theoretical and NMR, have been analyzed. The importance of these motions in terms of protein dynamics are revealed and envisioned by the normal mode analysis. Additional residues are proposed as candidates for further study of their potential promotional function.

Molecular dynamics and hybrid quantum mechanics/molecular mechanics have been used to investigate the mechanisms of ⁺ AdoMet methylation of protein-Lys-NH ₂ catalyzed by the lysine methyltransferase enzymes: histone lysine monomethyltransferase SET7/9, Rubisco large-subunit dimethyltransferase, viral histone lysine trimethyltransferase, and the Tyr245Phe mutation of SET7/9. At neutrality in aqueous solution, primary amines are protonated. The enzyme reacts with Lys-NH ₃ ⁺ and ⁺ AdoMet species to provide an Enz·Lys-NH ₃ ⁺ · ⁺ AdoMet complex. The close positioning of two positive charges lowers the pK _a of the Lys-NH ₃ ⁺ entity, a water channel appears, and the proton escapes to the aqueous solvent; then the reaction Enz·Lys-NH ₂ · ⁺ AdoMet → Enz·Lys-N(Me)H ₂ ⁺ ·AdoHcy occurs. Repeat of the sequence provides dimethylated lysine, and another repeat yields a trimethylated lysine. The sequence is halted at monomethylation when the conformation of the Enz·Lys-N(Me)H ₂ ⁺ · ⁺ AdoMet has the methyl positioned to block formation of a water channel. The sequence of reactions stops at dimethylation if the conformation of Enz·Lys-N(Me) ₂ H ⁺ · ⁺ AdoMet has a methyl in position, which forbids the formation of the water channel.

At neutral pH, oxidation of CH ₃ OH → CH ₂ O by an o -quinone requires general-base catalysis and the reaction is endothermic. The active-site –CO ₂ ⁻ groups of Glu-171 and Asp-297 (Glu-171–CO ₂ ⁻ and Asp-297–CO ₂ ⁻ ) have been considered as the required general base catalysts in the bacterial o -quinoprotein methanol dehydrogenase (MDH) reaction. Based on quantum mechanics/molecular mechanics (QM/MM) calculations, the free energy for MeOH reduction of o -PQQ when MeOH is hydrogen bonded to Glu-171–CO ₂ ⁻ and the crystal water (Wat1) is hydrogen bonded to Asp-297–CO ₂ ⁻ is Δ G ^‡ = 11.7 kcal/mol, which is comparable with the experimental value of 8.5 kcal/mol. The calculated Δ G ^‡ when MeOH is hydrogen bonded to Asp-297–CO ₂ ⁻ is >50 kcal/mol. The Asp-297–CO ₂ ⁻ ···Wat1 complex is very stable. Molecular dynamics (MD) simulations on MDH·PQQ·Wat1 complex in TIP3P water for 5 ns does not result in interchange of Asp-297–CO ₂ ⁻ bound Wat1 for a solvent water. Starting with Wat1 removed and MeOH hydrogen bonded to Asp-297–CO ₂ ⁻ , we find that MeOH returns to be hydrogen bonded to Glu-171–CO ₂ ⁻ and Asp-297–CO ₂ ⁻ coordinates to Ca ²⁺ during 3 ns simulation. The Asp-297–CO ₂ ⁻ ···Wat1 of reactant complex does play a crucial role in catalysis. By QM/MM calculation Δ G ^‡ = 1.1 kcal/mol for Asp-297–CO ₂ ⁻ general-base catalysis of Wat1 hydration of the immediate CH ₂ O product → CH ₂ (OH) ₂ . By this means, the endothermic oxidation-reduction reaction is pulled such that the overall conversion of MeOH to CH ₂ (OH) ₂ is exothermic.

We describe a quantum mechanics/molecular mechanics investigation of the guanidinoacetate methyltransferase catalyzed reaction, which shows that proton transfer from guanidinoacetate (GAA) to Asp-134 and methyl transfer from S-adenosyl-L-methionine (AdoMet) to GAA are concerted. By self-consistent-charge density functional tight binding/molecular mechanics, the bond lengths in the concerted mechanism's transition state are 1.26 A for both the OD1 (Asp-134)-H(E) (GAA) and H(E) (GAA)-N(E) (GAA) bonds, and 2.47 and 2.03 A for the S8 (AdoMet)-C9 (AdoMet) and C9 (AdoMet)-N(E) (GAA) bonds, respectively. The potential-energy barrier (DeltaE++) determined by single-point B3LYP/6-31+G*//MM is 18.9 kcal/mol. The contributions of the entropy (-TDeltaS++) and zero-point energy corrections Delta(ZPE)++ by normal mode analysis are 2.3 kcal/mol and -1.7 kcal/mol, respectively. Thus, the activation enthalpy of this concerted mechanism is predicted to be DeltaH++ = DeltaE++ plus Delta(ZPE)++ = 17.2 kcal/mol. The calculated free-energy barrier for the concerted mechanism is DeltaG++ = 19.5 kcal/mol, which is in excellent agreement with the value of 19.0 kcal/mol calculated from the experimental rate constant (3.8 +/- 0.2.min(-1)).

The mechanism of DNA cytosine-5-methylation catalyzed by the bacterial M.HhaI enzyme has been considered as a stepwise nucleophilic addition of Cys-81-S ⁻ to cytosine C6 followed by C5 nucleophilic replacement of the methyl of S -adenosyl- l -methionine to produce 5-methyl-6-Cys-81-S-5,6-dihydrocytosine. In this study, we show that the reaction is concerted from a series of energy calculations by using the quantum mechanical/molecular mechanical hybrid method. Deprotonation of 5-methyl-6-Cys-81-S-5,6-dihydrocytosine and expulsion of Cys-81-S ⁻ provides the product DNA 5-methylcytosine. A required base catalyst for this deprotonation is not available as a member of the active site structure. A water channel between the active site and bulk water allows entrance of solvent to the active site. Hydroxide at 10 ⁻⁷ mole fraction (pH = 7) is shown to be sufficient for the required catalysis. We also show that Glu-119-CO ₂ H can divert the reaction by protonating cytosine N3 when Cys-81-S ⁻ attacks cytosine, to form the 6-Cys-81-S-3-hydrocytosine. The reactants and 6-Cys-81-S-3-hydrocytosine product are in rapid equilibrium, and this explains the observed hydrogen exchange of cytosine with solvent.