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We studied proposed steps for the enzymatic formation of gamma-carboxyglutamic acid by density functional theory (DFT) quantum chemistry. Our results for one potentially feasible mechanism 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. The hydrated carbanion can then react with CO2 to form gamma-carboxyglutamic acid. Computations at the B3LYP/6-311G** level were used to determine the intermediates and transition states for the overall process. The activation free energy for the gas-phase path is 22 kcal/mol, with the rate-limiting step for the reaction being the attack of the carbanion on CO2. Additional solvation studies, however, indicate that the formation of the carbanion step can be competitive with the CO2 attack step in high-dielectric systems. We relate these computations to the entire vitamin K cycle in the blood coagulation cascade, which is essential for viability of vertebrates.

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.

An argument is presented that the spontaneous mutation rate, the core of evolution theory, may be dictated by the deuterium/hydrogen (D/H) abundance ratio. This argument is supported by quantum mechanical calculations of the zero-point energy reduction for DNA base pairs upon deuterium substitution for hydrogen and recent experiments that show that the rate of catalytic dsDNA unwinding is dependent on the stability of the dsDNA.

The accurate simulation of biologically active macromolecules faces serious limitations that originate in the treatment of electrostatics in the empirical force fields. The current use of "partial charges" is a significant source of errors, since these vary widely with different conformations. By contrast, the molecular electrostatic potential (MEP) obtained through the use of a distributed multipole moment description, has been shown to converge to the quantum MEP outside the van der Waals surface, when higher order multipoles are used. However, in spite of the considerable improvement to the representation of the electronic cloud, higher order multipoles are not part of current classical biomolecular force fields due to the excessive computational cost. In this paper we present an efficient formalism for the treatment of higher order multipoles in Cartesian tensor formalism. The Ewald "direct sum" is evaluated through a McMurchie-Davidson formalism [L. McMurchie and E. Davidson, J. Comput. Phys. 26, 218 (1978)]. The "reciprocal sum" has been implemented in three different ways: using an Ewald scheme, a particle mesh Ewald (PME) method, and a multigrid-based approach. We find that even though the use of the McMurchie-Davidson formalism considerably reduces the cost of the calculation with respect to the standard matrix implementation of multipole interactions, the calculation in direct space remains expensive. When most of the calculation is moved to reciprocal space via the PME method, the cost of a calculation where all multipolar interactions (up to hexadecapole-hexadecapole) are included is only about 8.5 times more expensive than a regular AMBER 7 [D. A. Pearlman et al., Comput. Phys. Commun. 91, 1 (1995)] implementation with only charge-charge interactions. The multigrid implementation is slower but shows very promising results for parallelization. It provides a natural way to interface with continuous, Gaussian-based electrostatics in the future. It is hoped that this new formalism will facilitate the systematic implementation of higher order multipoles in classical biomolecular force fields., ((c) 2004 American Institute of Physics)

EXTL2, an alpha1,4-N-acetylhexosaminyltransferase, catalyzes the transfer reaction of N-acetylglucosamine and N-acetylgalactosamine from the respective UDP-sugars to the non-reducing end of [glucuronic acid]beta1-3[galactose]beta1-O-naphthalenemethanol, an acceptor substrate analog of the natural common linker of various glycosylaminoglycans. We have solved the x-ray crystal structure of the catalytic domain of mouse EXTL2 in the apo-form and with donor substrates UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. In addition, a structure of the ternary complex with UDP and the acceptor substrate analog [glucuronic acid]beta1-3[galactose]beta1-O-naphthalenemethanol has been determined. These structures reveal three highly conserved residues, Asn-243, Asp-246, and Arg-293, located at the active site. Mutation of these residues greatly decreases the activity. In the ternary complex, an interaction exists between the beta-phosphate of the UDP leaving group and the acceptor hydroxyl of the substrate that may play a functional role in catalysis. These structures represent the first structures from the exostosin gene family and provide important insight into the mechanisms of alpha1,4-N-acetylhexosaminyl transfer in heparan biosynthesis.

The solution structure of mouse cytochrome P450 2A4 (CYP2A4), a monooxygenase of deoxysteroids, was obtained using homology modeling and molecular dynamics. The solvent-equilibrated CYP2A4 preserves the essential features of CYP450s. A comparison of the models CYP2A4 and CYP2A4 with testosterone bound CYP2A4/T illustrates the changes induced by the binding of the substrate. Experimental evidence links four amino acid residues to the catalytic activity, substrate specificity, and regioselectivity of this enzyme. Three of the four amino acids are found within contact distance of the testosterone substrate, and therefore may control the binding of the substrate through direct interaction. Remarkably, a water complex previously observed in x-ray crystal structure forms near the bulge in the central I helix that contains a conserved Thr. The properties of the I helix are computed in the context of the presence or absence of ligand.

Trocarin belongs to group D of prothrombin activators derived from snake venom of Tropidechis carinatus and is a rich non-hepatic source of Xa, the only known hepatic prothrombin activator. The structural and functional similarity with Xa makes trocarin an interesting target for exploring the structure-functional relationship with Xa. Herein we report a predicted complete three-dimensional all-atom structural model of trocarin equilibrated in explicit water using 4 ns of molecular dynamics simulation. The tertiary structure was modeled using the structure of human blood coagulation factor Xa. The conformational and structural features of trocarin are then compared with the X-ray crystal and solution simulation structures of human factor Xa. The modeled structure of trocarin has four individual domains (Gla, EGF1, EGF2 and SP) connected along the long axis with similar secondary structural elements to Xa. The simulations suggest that sodium ion binding in the serine protease domain is impaired in trocarin as compared to Xa. In contrast to Xa, for which the sodium ion forms an octahedral coordination network that brings two loop regions connecting four anti-parallel beta-sheets together, we do not find a similar pattern of network in trocarin. We observe that the difference in the binding pattern of sodium ion leads to a approximately 2-A "shrinkage" of the beta2 strand (B2), in comparison to human Xa, as marked by a shorter distance between 189Asp373 (S1-site residue) and 195Ser379 (active-site residue) in the B2 strand. We propose that these differences may be linked to the experimentally observed lower amidolytic activity of trocarin as compared to Xa.