The O2-dependent oxidation of urate catalyzed by urate oxidase has been examined in order to identify the immediate product of the enzymatic reaction. Specifically labeled [13C]urates were utilized as substrates, and the time courses were monitored by 13C NMR. On the basis of chemical shift values and 18O labeling, the product of the reaction was identified as 5-hydroxyisourate. This identification was substantiated by calculation of the 13C NMR spectrum of 5-hydroxyisourate using ab initio density functional theory methods. The predominant tautomers of urate and allantoin in aqueous solution were identified from 13C NMR titration data; the ionization behavior of urate and 5-hydroxyisourate were also examined by computational methods. The nonenzymatic pathway for production of allantoin from 5-hydroxyisourate was delineated; the reaction proceeds by the hydrolysis of the N1−C6 bond, followed by an unusual 1,2-carboxylate shift and decarboxylation to form allantoin.

The oxidation of urate catalyzed by soybean urate oxidase was studied under single-turnover conditions using stopped-flow absorbance and fluorescence spectrophotometry. Two discrete enzyme-bound intermediates were observed; the first intermediate to form had an absorbance maximum at 295 nm and was assigned to a urate dianion species; the second intermediate had an absorbance maximum at 298 nm and is believed to be urate hydroperoxide. These data are consistent with a catalytic mechanism that involves formation of urate hydroperoxide from O2 and the urate dianion, collapse of the peroxide to form dehydrourate, and hydration of dehydrourate to form the observed product, 5-hydroxyisourate. The rate of formation of the first intermediate was too fast to measure accurately at 20 degreesC; the second intermediate formed with a rate constant of 32 s-1 and decayed with a rate constant of 6.6 s-1. The product of the reaction, 5-hydroxyisourate, is fluorescent, and its release from the active site occurred with a rate constant of 31 s-1.

Hybrid quantum mechanics͞molecular mechanics calculations using Austin Model 1 system-specific parameters were performed to study the S N2 displacement reaction of chloride from 1,2-dichloroethane (DCE) by nucleophilic attack of the carboxylate of acetate in the gas phase and by Asp-124 in the active site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The activation barrier for nucleophilic attack of acetate on DCE depends greatly on the reactants having a geometry resembling that in the enzyme or an optimized gas-phase structure. It was found in the gas-phase calculations that the activation barrier is 9 kcal͞mol lower when dihedral constraints are used to restrict the carboxylate nucleophile geometry to that in the enzyme relative to the geometries for the reactants without dihedral constraints. The calculated quantum mechanics͞molecular mechanics activation barriers for the enzymatic reaction are 16.2 and 19.4 kcal͞mol when the geometry of the reactants is in a near attack conformer from molecular dynamics and in a conformer similar to the crystal structure (DCE is gauche), respectively. This haloalkane dehalogenase lowers the activation barrier for dehalogenation of DCE by 2-4 kcal͞mol relative to the single point energies of the enzyme's quantum mechanics atoms in the gas phase. S N2 displacements of this sort in water are infinitely slower than in the gas phase. The modest lowering of the activation barrier by the enzyme relative to the reaction in the gas phase is consistent with mutation experiments.H aloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds in aliphatic and aromatic halogenated compounds. The haloalkane dehalogenase from the nitrogen-fixing hydrogen bacterium Xanthobacter autotrophicus GJ10 (DhlA) prefers 1,2-dichloroethane (DCE) as substrate and converts it to 2-chloroethanol and chloride (1). A catalytic triad consisting of Asp-124, His-289, and Asp-260 is the central residue in the dehalogenation reaction (Scheme 1). On binding DCE in the predominantly hydrophobic active site, it undergoes S N 2 displacement of chloride by nucleophilic attack of Asp-124-COO Ϫ to form an ester intermediate at the rate of 50 Ϯ 10 s Ϫ1(1, 2). The ester intermediate is subsequently hydrolyzed by an activated water molecule. The dyad of His-289 and Asp-260 is thought to be responsible for activating the water molecule (3). This enzyme functions most efficiently at pH 8.2, likely because the imidazole NE2 of His-289 needs to be unprotonated for the hydrolysis reaction to proceed. Dehalogenases are of great interest because they are able to react with halogenated molecules under mild conditions (4). Many halogenated molecules are pollutants, and bioremediation is a highly desirable method for removing these harmful molecules from the environment. Dehalogenases have not existed in nature for a long time andhave not yet evolved into optimal enzymes. For example, the catalytic efficiency for DhlA is 4,550 M Ϫ1 ⅐s Ϫ1 for DCE (5), as compared with 5.6 ϫ 10 7 M Ϫ1 ⅐s Ϫ1for...

The parameters for the OPLS-AA potential energy function have been extended to include some functional groups that are present in macrocyclic polyketides. Existing OPLS-AA torsional parameters for alkanes, alcohols, ethers, hemiacetals, esters, and ketoamides were improved based on MP2/aug-cc-pVTZ and MP2/aug-cc-pVDZ calculations. Nonbonded parameters for the sp(3) carbon and oxygen atoms were refined using Monte Carlo simulations of bulk liquids. The resulting force field predicts conformer energies and torsional barriers of alkanes, alcohols, ethers, and hemiacetals with an overall RMS deviation of 0.40 kcal/mol as compared to reference data. Densities of 19 bulk liquids are predicted with an average error of 1.1%, and heats of vaporization are reproduced within 2.4% of experimental values. The force field was used to perform conformational analysis of smaller analogs of the macrocyclic polyketide drug FK506. Structures that adopted low-energy conformations similar to that of bound FK506 were identified. The results show that a linker of four ketide units constitutes the shortest effector domain that allows binding of the ketide drugs to FKBP proteins. It is proposed that the exact chemical makeup of the effector domain has little influence on the conformational preference of tetraketides.