Theoretical insights into the protonation states of active site cysteine and citrullination mechanism of Porphyromonas gingivalis peptidylarginine deiminase

Porphyromonas gingivalis peptidylarginine deiminase (PPAD) catalyzes the citrullination of peptidylarginine, which plays a critical role in the rheumatoid arthritis (RA) and gene regulation. For a better understanding of citrullination mechanism of PPAD, it is required to establish the protonation states of active site cysteine, which is still a controversial issue for the members of guanidino‐group‐modifying enzyme superfamily. In this work, we first explored the transformation between the two states: State N (both C351 and H236 are neutral) and State I (both residues exist as a thiolate–imidazolium ion pair), and then investigated the citrullination reaction of peptidylarginine, using a combined QM/MM approach. State N is calculated to be more stable than State I by 8.46 kcal/mol, and State N can transform to State I via two steps of substrate‐assisted proton transfer. Citrullination of the peptidylarginine contains deamination and hydrolysis. Starting from State N, the deamination reaction corresponds to an energy barrier of 18.82 kcal/mol. The deprotonated C351 initiates the nucleophilic attack to the substrate, which is the key step for deamination reaction. The hydrolysis reaction contains two chemical steps. Both the deprotonated D238 and H236 can act as the bases to activate the hydrolytic water, which correspond to similar energy barriers (～17 kcal/mol). On the basis of our calculations, C351, D238, and H236 constitute a catalytic triad, and their protonation states are critical for both the deamination and hydrolysis processes. In view of the sequence similarity, these findings may be shared with human PAD1–PAD4 and other guanidino‐group‐modifying enzymes. Proteins 2017; 85:1518–1528. © 2017 Wiley Periodicals, Inc.     

Hexahydrated Mg2+ Binding and Outer-Shell Dehydration on RNA Surface

The interaction between metal ions, especially Mg²⁺ ions, and RNA plays a critical role in RNA folding. Upon binding to RNA, a metal ion that is fully hydrated in bulk solvent can become dehydrated. Here we use molecular dynamics simulation to investigate the dehydration of bound hexahydrated Mg²⁺ ions. We find that a hydrated Mg²⁺ ion in the RNA groove region can involve significant dehydration in the outer hydration shell. The first or innermost hydration shell of the Mg²⁺ ion, however, is retained during the simulation because of the strong ion-water electrostatic attraction. As a result, water-mediated hydrogen bonding remains an important form for Mg²⁺-RNA interaction. Analysis for ions at different binding sites shows that the most pronounced water deficiency relative to the fully hydrated state occurs at a radial distance of around 11 Å from the center of the ion. Based on the independent 200 ns molecular dynamics simulations for three different RNA structures (Protein Data Bank: 1TRA, 2TPK, and 437D), we find that Mg²⁺ ions overwhelmingly dominate over monovalent ions such as Na⁺ and K⁺ in ion-RNA binding. Furthermore, application of the free energy perturbation method leads to a quantitative relationship between the Mg²⁺ dehydration free energy and the local structural environment. We find that $\text{ΔΔ}{G}_{\text{hyd}},$ the change of the Mg²⁺ hydration free energy upon binding to RNA, varies linearly with the inverse distance between the Mg²⁺ ion and the nearby nonbridging oxygen atoms of the phosphate groups, and $\text{ΔΔ}{G}_{\text{hyd}}$ can reach ?2.0 kcal/mol and ?3.0 kcal/mol for an Mg²⁺ ion bound to the surface and to the groove interior, respectively. In addition, the computation results in an analytical formula for the hydration ratio as a function of the average inverse Mg²⁺-O distance. The results here might be useful for further quantitative investigations of ion-RNA interactions in RNA folding.     

Structural analysis of the active site architecture of the VapC toxin from Shigella flexneri

The VapC toxin from the Shigella flexneri 2a virulence plasmid pMYSH6000 belongs to the PIN domain protein family, which is characterized by a conserved fold with low amino acid sequence conservation. The toxin is a bona fide Mg²⁺‐dependent ribonuclease and has been shown to target initiator tRNA^fMet in vivo. Here, we present crystal structures of active site catalytic triad mutants D7A, D7N, and D98N of the VapC toxin in absence of antitoxin. In all structures, as well as in solution, VapC forms a dimer. In the D98N structure, a Hepes molecule occupies both active sites of the dimer and comparison with the structure of RNase H bound to a DNA/RNA hybrid suggests that the Hepes molecule mimics the position of an RNA nucleotide in the VapC active site. Proteins 2016; 84:892–899. © 2016 Wiley Periodicals, Inc.     

Thermodynamic examination of the pyrophosphate sensor helix in the thiamine pyrophosphate riboswitch

Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 × 2 bulged RNA and one 2 × 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 × 2 bulge (C65 and G66). The RNA WT construct was among the most stable (ΔG°₃₇ = −7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A•G mismatch of the bulge decreases the stability of the construct ∼0.5–1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA–Mg²⁺ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/mol relative to 1 M salt. Formation of a single A+•C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+•C mismatch base pairs together contribute about 2 kcal/mol.     

Computational delineation of the catalytic step of a high‐fidelity DNA polymerase

The Bacillus fragment, belonging to a class of high‐fidelity polymerases, demonstrates high processivity (adding ～115 bases per DNA binding event) and exceptional accuracy (1 error in 10⁶ nucleotide incorporations) during DNA replication. We present analysis of structural rearrangements and energetics just before and during the chemical step (phosphodiester bond formation) using a combination of classical molecular dynamics, mixed quantum mechanics molecular mechanics simulations, and free energy computations. We find that the reaction is associative, proceeding via the two‐metal‐ion mechanism, and requiring the proton on the terminal primer O3′ to transfer to the pyrophosphate tail of the incoming nucleotide before the formation of the pentacovalent transition state. Different protonation states for key active site residues direct the system to alternative pathways of catalysis and we estimate a free energy barrier of ～12 kcal/mol for the chemical step. We propose that the protonation of a highly conserved catalytic aspartic acid residue is essential for the high processivity demonstrated by the enzyme and suggest that global motions could be part of the reaction free energy landscape.     

Equilibrium constants under physiological conditions for the reactions of the nonphosphorylated pathway of L-serine biosynthesis

The observed equilibrium constants (K_obs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate^3? + H₂O → d-glycerate^? + HPO₄^2?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate^? + NAD⁺ → NADH + hydroxypyruvate^? + H⁺; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate^? + l-H · alanine^± → pyruvate^? + l-H · serine^±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 10³m [ΔG⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)]([H₂0] = 1). Values of K_obs for this reaction at 38 °C, [K⁺] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 10³m (free [Mg²⁺] = 0), 3.23 × 10³m (free [Mg²⁺] = 10^?3m), and 2.32 × 10³m (free [Mg²⁺] = 10^?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10^?13m (38 °C, I = 0.25 M) [ΔG⁰ = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH⁰ = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG⁰ = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH⁰ = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of K_obs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K⁺] = 0.2 m, [Mg²⁺] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate^? + ATP^4? → d-2-phosphoglycerate^3? + ADP^3? + H⁺, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for K_obs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg²⁺] = 0) and 50.8 (free [Mg²⁺] = 10^?3m).     

A QM/MM study of the reaction mechanism of (R)‐hydroxynitrile lyases from Arabidopsis thaliana (AtHNL)

Hydroxynitrile lyases (HNLs) catalyze the conversion of chiral cyanohydrins to hydrocyanic acid (HCN) and aldehyde or ketone. Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) is the first R‐selective HNL enzyme containing an α/β‐hydrolases fold. In this article, the catalytic mechanism of AtHNL was theoretically studied by using QM/MM approach based on the recently obtained crystal structure in 2012. Two computational models were constructed, and two possible reaction pathways were considered. In Path A, the calculation results indicate that the proton transfer from the hydroxyl group of cyanohydrin occurs firstly, and then the cleavage of C1‐C2 bond and the rotation of the generated cyanide ion (CN^?) follow, afterwards, CN^? abstracts a proton from His236 via Ser81. The C1‐C2 bond cleavage and the protonation of CN^? correspond to comparable free energy barriers (12.1 vs. 12.2 kcal mol^?1), suggesting that both of the two processes contribute a lot to rate‐limiting. In Path B, the deprotonation of the hydroxyl group of cyanohydrin and the cleavage of C1‐C2 bond take place in a concerted manner, which corresponds to the highest free energy barrier of 13.2 kcal mol^?1. The free energy barriers of Path A and B are very similar and basically agree well with the experimental value of HbHNL, a similar enzyme of AtHNL. Therefore, both of the two pathways are possible. In the reaction, the catalytic triad (His236, Ser81, and Asp208) acts as the general acid/base, and the generated CN^? is stabilized by the hydroxyl group of Ser81 and the main‐chain NH‐groups of Ala13 and Phe82. Proteins 2015; 83:66–77. © 2014 Wiley Periodicals, Inc.     

Alterations in phospholipid-dependent (Na++K+)-ATPase activity due to lipid fluidity: Effects of cholesterol and Mg2+

The (Na⁺+K⁺)-activated, Mg²⁺-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na⁺+K⁺)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg²⁺: ATP mol ratios of approximately 1 and a K_m value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg²⁺: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (E_a) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the E_a below this was very high (> 100 kcal/mol).Incresed Mg²⁺ concentrations at Mg²⁺: ATP ratios in excess of 1:1 inhibited the (Na⁺+K⁺)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na⁺+K⁺)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and E_a values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg²⁺-ATPase normally showed a linear Arrhenius plot with an E_a of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg²⁺-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the K_m for ATP.Since both of cholesterol and Mg²⁺ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45°C in the absence and presence of Mg²⁺. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. ΔH⁰ and ΔS⁰ for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, ΔH⁰ ≈ 15–16 kcal/mol (63–67 kJ/mol) and ΔS⁰ ≈ 50–57 cal/K per mol (209–239 J/K per mol). The presence of Mg²⁺ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg²⁺, the ΔH⁰ and ΔS⁰ values are larger and temperature-dependent, with ΔH⁰ ≈ 22 kcal/mol (92 kJ/mol) and ΔS⁰ ≈ 72 cal/K per mol (approx. 300 J/K per mol) at 25°C, and ΔC_p⁰ 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

Comparative studies of the catalytic mechanisms of two chorismatases: CH‐fkbo and CH‐Hyg5

Chorismatase is an important enzyme involved in Shikimate pathway, which catalyzes the conversion of chorismate into pyruvate and (dihydro)‐benzoic acid derivatives. According to the outcomes of catalytic reactions, chorismatases can be divided into three subfamilies: CH‐Fkbo, CH‐Hyg5 and CH‐XanB2. Recently, the crystal structures of CH‐Fkbo and CH‐Hyg5 from Streptomyces hygroscopicus have been successfully obtained, allowing us to perform QM/MM calculations to explore the reaction details. Our calculation results support the proposal that CH‐Fkbo and CH‐Hyg5 employ different catalytic mechanisms and gave the mechanistic details. Fkbo follows a typical hydrolytic mechanism, which contains three consecutive steps, including the protonation step of the methylene group of substrate, the nucleophilic attack of the resulted carbocation by activated water and cleavage of C2′‐O8 bond of tetrahedral intermediate (hemiketal). The protonation of methylene group and the C2′‐O8 cleavage correspond to similar energy barriers (26.5 and 24.8 kcal/mol), suggesting both steps to be rate‐limiting. Whereas Hyg5 employs an intramolecular mechanism, in which the oxygen from C4 migrates to C3 via an arene oxide intermediate. The first step of Hyg5, which corresponds to the concerted protonation of methylene group and the cleavage of C3‐O8, is calculated to be rate‐limiting with an energy barrier of 26.3 kcal/mol. The nonconserved active site residue G240^Hyg5 (or A244^Fkb°) is suggested to be responsible for leading to different reaction mechanism in CH‐Fkbo and CH‐Hyg5. During the catalytic reaction, residue C327 plays an important role in directing the product selectivity in Hyg5 enzyme. Proteins 2017; 85:1146–1158. © 2017 Wiley Periodicals, Inc.     

Hydration of carbon dioxide by carbonic anhydrase: internal proton transfer of Zn2+-bound HCO3-

Proton transfer within HCO3- has been examined under various conditions through molecular orbital methods: partial retention of diatomic differential overlap and 4-31G self-consistent field programs. These conditions include the absence or presence of Zn2+, Zn2+(NH3)3, or a water ligand on Zn2+. In addition, 4-31G+ and some MP2/4-31G results are obtained. The use of Be2+ to simulate Zn2+ reproduces reaction pathways and energy barriers, except for marginal cases. The barrier of 35.6 kcal/mol for direct internal proton transfer is reduced to 3.5 kcal/mol when one water molecule, not bound to Zn2+, is included for proton relay and to 1.4 kcal/mol when two such water molecules are included. In the enzyme, either Thr-199 or solvent molecules could perform this relay function. Our results favor this facilitated proton transfer over a mechanism in which Zn2+-bound OH- attacks CO2, a bidentate intermediate forms, and the OH moiety of the resulting HCO3- dissociates from Zn2+, thus leaving one of the oxygens of the original CO2 as a ligand to Zn2+.     

Thermodynamic and structural properties of pentamer DNA.DNA, RNA.RNA, and DNA.RNA duplexes of identical sequence

Four pentamers with the general sequence 5'CU(T)GU(T)G/5'CACAG have been prepared by chemical synthesis in order to generate duplex structures with common sequences. The four duplexes studied include the DNA.DNA duplex (5'dCACAG/5'dCTGTG) and the RNA.RNA duplex (5'rCUGUG/5'rCACAG) as well as the two corresponding DNA.RNA heteroduplexes (5'rCUGUG/5'dCACAG and 5'CACAG/5'dCTGTG). The measured entropy, enthalpy, and free energy changes upon melting are reported for each pentamer and compared to the predicted values where possible. Results show that the two DNA.RNA heteroduplexes are destabilized (delta G degrees 25 = -4.2 +/- 0.4 kcal/mol) relative to either the DNA.DNA duplex (delta G degrees 25 = -4.8 +/- 0.5 kcal/mol) or the RNA.RNA duplex (delta G degrees 25 = -5.8 +/- 0.6 kcal/mol). Circular dichroism spectra indicate that the RNA and the two heteroduplexes adopt an A-form conformation, while the DNA conformation is B-form. Imino proton NMR spectra also show that the heteroduplex structures resemble the RNA.RNA duplex.     

Interactions of the acid and base catalysts on staphylococcal nuclease as studied in a double mutant.

The mechanism of the phosphodiesterase reaction catalyzed by staphylococcal nuclease is believed to involve concerted general acid-base catalysis by Arg-87 and Glu-43. The mutual interactions of Arg-87 and Glu-43 were investigated by comparing kinetic and thermodynamic properties of the single mutant enzymes E43S (Glu-43 to Ser) and R87G (Arg-87 to Gly) with those of the double mutant, E43S + R87G, in which both the basic and acidic functions have been inactivated. Denaturation studies with guanidinium chloride, CD, and 600-MHz 1D and 2D proton NMR spectra, indicate all enzyme forms to be predominantly folded in absence of the denaturant and reveal small antagonistic effects of the E43S and R87G mutations on the stability and structure of the wild-type enzyme. The free energies of binding of the divalent cation activator Ca2+, the inhibitor Mn2+, and the substrate analogue 3',5'-pdTp show simple additive effects of the two mutations in the double mutant, indicating that Arg-87 and Glu-43 act independently to facilitate the binding of divalent cations and of 3',5'-pdTP by the wild-type enzyme. The free energies of binding of the substrate, 5'-pdTdA, both in binary E-S and in active ternary E-Ca(2+)-S complexes, show synergistic effects of the two mutations, suggesting that Arg-87 and Glu-43 interact anticooperatively in binding the substrate, possibly straining the substrate by 1.6 kcal/mol in the wild-type enzyme. The large free energy barriers to Vmax introduced by the R87G mutation (delta G1 = 6.5 kcal/mol) and by the E43S mutation (delta G2 = 5.0 kcal/mol) are partially additive in the double mutant (delta G1+2 = 8.1 kcal/mol). These partially additive effects on Vmax are most simply explained by a cooperative component to transition state binding by Arg-87 and Glu-43 of -3.4 kcal/mol. The combination of anticooperative, cooperative, and noncooperative effects of Arg-87 and Glu-43 together lower the kinetic barrier to catalysis by 8.1 kcal/mol.     

Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA

RNA structures contain many bulges and loops that are expected to be sites for inter- and intra-molecular interactions. Nucleotides in the bulge are expected to influence the structure and recognition of RNA. The same stability is assigned to all trinucleotide bulged RNA in the current secondary structure prediction models. In this study thermal denaturation experiments were performed on four trinucleotide bulged RNA, in the context of HIV-1 TAR RNA, to determine whether the bulge sequence affects RNA stability and its divalent ion interactions. Cytosine-rich bulged RNA were more stable than uracil-rich bulged RNA in 1 M KCl. Interactions of divalent ions were more favorable with uracil-rich bulged RNA by ~2 kcal/mol over cytosine-rich bulged RNA. The UCU-TAR RNA (wild type) is stabilized by 1.7 kcal/mol in 9.5 mM Ca²⁺ as compared with 1 M KCl, whereas no additional gain in stability is measured for CCC-TAR RNA. These results have implications for base substitution experiments traditionally employed to identify metal ion binding sites. To our knowledge, this is the first systematic study to quantify the effect of small sequence changes on RNA stability upon interactions with divalent ions.     

Catalytic mechanism and product specificity of rubisco large subunit methyltransferase: QM/MM and MD investigations

Molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations have been carried out in an investigation of Rubisco large subunit methyltransferase (LSMT). It was found that the appearance of a water channel is required for the stepwise methylation by S-adenosylmethionine (AdoMet). The water channel appears in the presence of AdoMet (LSMT.Lys-NH3+.AdoMet), but is not present immediately after methyl transfer (LSMT.Lys-N(Me)H2+.AdoHcy). The water channel allows proton dissociation from both LSMT.AdoMet.Lys-NH3+ and LSMT.AdoMet.Lys-N(Me)H2+. The water channel does not appear for proton dissociation from LSMT.AdoMet.Lys-N(Me)2H+, and a third methyl transfer does not occur. By QM/MM, the calculated free energy barrier of the first methyl transfer reaction catalyzed by LSMT (Lys-NH2 + AdoMet --> Lys-N(Me)H2+ + AdoHcy) is DeltaG++ = 22.8 +/- 3.3 kcal/mol. This DeltaG++ is in remarkable agreement with the value 23.0 kcal/mol calculated from the experimental rate constant (6.2 x 10-5 s-1). The calculated DeltaG++ of the second methyl transfer reaction (AdoMet + Lys-N(Me)H --> AdoHcy + Lys-N(Me)2H+) at the QM/MM level is 20.5 +/- 3.6 kcal/mol, which is in agreement with the value 22.0 kcal/mol calculated from the experimental rate constant (2.5 x 10-4 s-1). The third methyl transfer (Lys-N(Me)2 + AdoMet --> Lys-N(Me)3+ + AdoHcy) is associated with an allowed DeltaG++ of 25.9 +/- 3.2 kcal/mol. However, this reaction does not occur because a water channel does not form to allow the proton dissociation of Lys-N(Me)2H+. Future studies will determine whether the product specificity of lysine (mono, di, and tri) methyltransferases is determined by the formation of water channels.     

Hydration of CO2 by carbonic anhydrase: intramolecular proton transfer between Zn2+-bound H2O and histidine 64 in human carbonic anhydrase II

The energy barrier for the intramolecular proton transfer between zinc-bound water and His 64 in the active site of human carbonic anhydrase II (HCA II) has been studied at the partial retention of diatomic differential overlap (PRDDO) level. The most important stabilizing factor for the intramolecular proton transfer is the zinc ion, which lowers the pKa of zinc-bound water and electrostatically repels the proton. The energy barrier of 127.5 kcal/mol for proton transfer between a water dimer is completely removed in the presence of the zinc ion. The zinc ligands, which donate electrons to the zinc ion, raise the barrier slightly to 34 kcal/mol for a 4-coordinated zinc complex including three imidazole ligands from His 94, His 96, and His 119 and to 54 kcal/mol for the 5-coordinated zinc complex including the fifth water ligand. A few model calculations indicate that these energy barriers are expected to be reduced to within experimental range (approximately 10 kcal/mol) when large basis set, correlation energies, and molecular dynamics are considered. The proton-transfer group, which functions as proton receiver in the intramolecular proton transfer, helps to attract the proton; and the partially ordered active site water molecules are important for proton relay function.     

Role of AP‐endonuclease (Ape1) active site residues in stabilization of the reactant enzyme‐DNA complex

Apurinic/apyrimidinic endonuclease 1 (Ape1) is an important metal‐dependent enzyme in the base excision repair mechanism, responsible for the backbone cleavage of abasic DNA through a phosphate hydrolysis reaction. Molecular dynamics simulations of Ape1 complexed to its substrate DNA performed for models containing 1 or 2 Mg²⁺‐ions as cofactor located at different positions show a complex with 1 metal ion bound on the leaving group site of the scissile phosphate to be the most likely reaction‐competent conformation. Active‐site residue His309 is found to be protonated based on pKa calculations and the higher conformational stability of the Ape1‐DNA substrate complex compared to scenarios with neutral His309. Simulations of the D210N mutant further support the prevalence of protonated His309 and strongly suggest Asp210 as the general base for proton acceptance by a nucleophilic water molecule.     

Substitution of an essential adenine in the U1A-RNA complex with a non-polar isostere

The RNA recognition motif (RRM) binds to single-stranded RNA target sites of diverse sequences and structures. A conserved mode of base recognition by the RRM involves the simultaneous formation of a network of hydrogen bonds with the base functional groups and a stacking interaction between the base and a highly conserved aromatic amino acid. We have investigated the energetic contribution of the functional groups involved in the recognition of an essential adenine, A6, in stem–loop 2 of U1 snRNA by the N-terminal RRM of the U1A protein. Previously, we found that elimination of individual hydrogen bond donors and acceptors on A6 destabilized the complex by 0.8–1.9 kcal/mol, while mutation of the aromatic amino acid (Phe56) that stacks with A6 to Ala destabilized the complex by 5.5 kcal/mol. Here we continue to probe the contribution of A6 to complex stability through mutation of both the RNA and protein. We have removed two hydrogen-bonding functional groups by introducing a U1A mutation, Ser91Ala, and replacing A6 with tubercidin, purine, or 1-deazaadenine. We find that the complex is destabilized an additional 1.2–2.6 kcal/mol by the elimination of the second hydrogen bond donor or acceptor. Surprisingly, deletion of all of the functional groups involved in hydrogen bonds with the U1A protein by substituting adenine with 4-methylindole reduced the binding free energy by only 2.0 kcal/mol. Experiments with U1A proteins containing mutations of Phe56 suggested that improved stacking interactions due to the greater hydrophobicity of 4-methylindole than adenine may be partly responsible for the small destabilization of the complex upon substitution of 4-methylindole for A6. The data imply that hydrophobic interactions can compensate energetically for the disruption of the complex hydrogen-bonding network between nucleotide and protein.