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Based on a recent ternary complex crystal structure of human DNA polymerase [beta] with a G:A mismatch in the active site, we carried out a theoretical investigation of the catalytic mechanism of incorrect nucleotide incorporation using molecular dynamics simulation, quantum mechanics, combined quantum mechanics, and molecular mechanics methods. A two-stage mechanism is proposed with a nonreactive active-site structural rearrangement prechemistry step occurring before the nucleotidyl transfer reaction. The free energy required for formation of the prechemistry state is found to be the major factor contributing to the decrease in the rate of incorrect nucleotide incorporation compared with correct insertion and therefore to fidelity enhancement. Hence, the transition state and reaction barrier for phosphodiester bond formation after the prechemistry state are similar to that for correct insertion reaction. Key residues that provide electrostatic stabilization of the transition state are identified. combined quantum mechanics and molecular mechanics | incorrect nucleotide incorporation | nucleotidyl transfer | two-stage mechanism

Density functional theory (DFT) quantum chemical study was carried out to examine steps for the enzymatic formation of [gamma]-carboxyglutamic acid. The results show that a vitamin K alkoxide intermediate can abstract a proton from glutamic acid at the [gamma]-carbon to form a carbanion and vitamin K epoxide which then reacts with C[O.sub.2] to form [gamma]-carboxyglutamic acid.

X-ray crystallographic structures of human DNA polymerase [beta] with nonhydrolyzable analogs containing all atoms in the active site required for catalysis provide a secure starting point for a theoretical analysis (quantum mechanics/molecular mechanics) of the mechanism of chemistry without biasing of modeling assumptions as required in previous studies. These structures provide the basis for a detailed quantum mechanics/molecular mechanics study of the path for the complete transfer of a monophosphate nucleoside donor to the sugar acceptor in the active site. The reaction is largely associative with the main energetic step preceded by proton transfer from the terminal primer deoxyribose O3' to Asp-256. The key residues that provide electrostatic stabilization of the transition state are identified and compared with those identified by mutational studies. quantum mechanics | molecular mechanics | reaction mechanism

A study was conducted to re-evaluate the trans-cis isomerization of proline 22 (Pro22) in bovine prothrombin residues 1-45 where molecular dynamics simulation methods were used to estimate equilibrated solution conformation of bovine prothrombin in both cis and trans forms. Observations suggest that if isomerization of Pro22 is required for phopholipid binding, the isomerization may occur without a structural consequence.

The function of gamma-carboxyglutamic acid (Gla) in the calcium-induced structural transition of conantokin G was examined. Conantokin G is a Gla-containing conotoxin isolated from the venom of the marine cone snail conus geographus. The three-dimensional structure of the conantokin G/Ca2+ complex was determined and compared to the high-resolution structure of conantokin G in the absence of metal ions. Findings showed that upon binding of Ca2+ to Gla, conantokin G undergoes a conformation transition from a distorted curvilinear 3(sub 10) helix to a linear alpha-helix.

The role of the side-chain size of residues 117, 209 and 481 in murine P450 (mP450) 2a4/2a5 system hydroxylates DHEA was analyzed by bacterial expression and site-directed mutagenesis of mP450 2a5 from wild Mus minutoides. Analysis of the effects of hydrophobic amino acid side-chain size on P450 activity indicated the presence of a reciprocal size-effect relationship that affects the specific activities of P role of the residue's size in defining P450 activity. Furthermore, the size of amino acid side chains altered the regio- and stereospecificities of steroid hydroxylase activity in the P450s.

Genetic algorithm (GA) was used to identify the calcium binding sites of the gamma-carboxylic acid domain of the coagulation factor IX. Molecular dynamics simulation was also performed using the most frequently occurring low-energy structure obtained by GA as the starting structure for the molecular dynamics refinement. The findings confirmed that calcium ions induced structural changes during the simulation experiment, yielding a more compact structure.

Site-directed mutagenesis experiments are performed to study the functional role of gamma-carboxyglutamic acid residue (GLA) 21 of the human factor IX during its mutation into aspartic acid. Results reveal that the mutation of GLA 21 into aspartic acid disrupts function in the N-terminal region of the GLA domain without affecting the structure of the C-terminal GLA domain region.

An ab initio quantum mechanical model for the catalytic mechanism of the HIV-1 protease is presented. The model, composed of a formate/formic acid pair, a structurally important water molecule and a formamide molecule, has a catalytic mechanism composed of five steps, including two transition states separated by an intermediate step and two steps related to product release. It is expected to prove valuable in understanding further the HIV-1 protease catalytic mechanism.

The carboxy-terminal halves of the p450 heme-thiolate oxygenases determine the regiospecificity of androstenedione (AD) hydroxylase activity, through the Val/Ala mutation at position 481. Mouse P450 2a-4 and 2a-5 mutations were tested to explore AD hydroxylase activity regiospecificity and found that the larger the hydrophobic amino acid at position 481 the higher the AD 15-alpha hydroxylase activity regiospecificity. Modeling of p450 101A indicates that the residue 481 affects the AD hydroxylase activity through a position near the steroid molecule.

Accurate predictions of molecular acidity using ab initio and density functional approaches are still a daunting task. Using electronic and reactivity properties, one can quantitatively estimate pKa values of acids. In a recent paper [S. B. Liu and L. G. Pedersen, J. Phys. Chem. A 113, 3648 (2009)], we employed the molecular electrostatic potential (MEP) on the nucleus and the sum of valence natural atomic orbital (NAO) energies for the purpose. In this work, we reformulate these relationships on the basis of conceptual density functional theory and compare the results with those from the thermodynamic cycle method. We show that MEP and NAO properties of the dissociating proton of an acid should satisfy the same relationships with experimental pKa data. We employ 27 main groups and first to third row transition metal-water complexes as illustrative examples to numerically verify the validity of these strong linear correlations. Results also show that the accuracy of our approach and that of the conventional method through the thermodynamic cycle are statistically similar. [ABSTRACT FROM AUTHOR]