Catalyzed decomposition of urea. Molecular dynamics simulations of the binding of urea to urease

We present the results of molecular dynamics simulations on the urea/urease system. The starting structure was prepared from the 2.0 A crystal structure of Benini et al. [(1999) Struct. Folding Des. 7, 205-216] of DAP-inhibited urease (PDB code ), and the trimeric structure (2479 residues) resulted in 180K atoms after solvation by water. The force field parameters were derived using the bonded model approach described by Hoops et al. [(1991) J. Am. Chem. Soc. 113, 8262-8270]. Three different systems were analyzed, each one modeling a different protonation pattern for the His320 and His219 residues. In each case, the three monomers of urease have been analyzed separately. The time-averaged structures observed in the three monomers suggest that urease could follow two different competitive mechanisms. A "protein-assisted proton transfer" mechanism points to Asp221 as crucial for catalysis. An "Asp-mediated proton transfer" involves the transfer of a proton from the bridging OH to an NH2 moiety of urea, assisted by Asp360 in the active site. The impact of the simulation results on our understanding of urease catalysis is discussed in detail.     

Urease-catalyzed urea synthesis.

The equilibrium of hydrolytic reactions can be shifted toward condensation by carrying out the reaction at low water concentration. The rate and yield of urease-catalyzed urea synthesis from (NH₄)₂CO₃ or NH₄HCO₃ has been examined as a function of water concentration (in mixtures with organic solvents), substrate and H⁺ concentration, and polarity of the nonaqueous component of the solvent. Similar effects of organic solvents are observed on the reaction rate in both directions; the results suggest that at least in some conditions the reaction proceeds through nonenzymically formed carbamate. The equilibrium concentration of urea, in 50% ($\text{v}\text{v}$) water, varies over 10-fold, depending on the nature of the nonaqueous component of the solvent; nonhydroxylic solvents such as acetone given the highest yield. Solubility measurements suggest that the interactions of the solvent mixtures with (NH₄)₂CO₃ (or carbamate), rather than urea, are responsible for the variations in urea yield. Activities of water and the ionic components of the equilibrium are strongly influenced by the nature of the nonaqueous component of the solvent, as well as its concentration.     

Thermodynamics of the denaturation of pepsinogen by urea.

The denaturation of swine pepsinogen has been studied as a function of urea concentration, pH, and temperature. The unfolding of the protein by urea has been found to be fully reversible under different conditions of pH, temperature, and denaturant concentration. Kinetic experiments have shown that the transition shows two-state behavior at 25 degrees C in the pH range 6-8 covered in this study. Analysis of the equilibrium data obtained at 25 degrees C according to Tanford (Tanford, C. (1970), Adv. Protein Chem. 24, 1) and Pace (Pace, N.C. (1975), Crit. Rev. Biochem. 3, 1) leads to the conclusion that the free energy of stabilization of native pepsinogen, relative to the denatured state, under physiological conditions, is only 6-12 kcal mol-1. The temperature dependence of the equilibrium constant for the unfolding of pepsinogen by urea in the range 20-50 degrees C at pH 8.0 can be described by assigning the following values of thermodynamic parameters for the denaturation at 25 degrees C: deltaH=31.5 kcal mol-1; deltaS=105 cal deg-1 mol-1; and deltaCp=5215 cal deg-1 mol-1.     

Electrophoretic analysis of the unfolding of proteins by urea.

The unfolding of several proteins by urea has been followed by electrophoresis of a band of protein through a slab gel of polyacrylamide in which there was a gradient of urea concentration perpendicular to the direction of electrophoresis. Unfolding was invariably manifested by a marked reduction of mobility, presumably due to molecular sieving of the expanded polypeptide chain by the polyacrylamide gel. The procedure provides a continuous two-dimensional pattern of the effect of urea on the shape of the protein and is especially sensitive to microheterogeneity of the protein.Experiments with pancreatic trypsin inhibitor, ribonuclease, lysozyme, chymotrypsin, chymotrypsinogen, staphylococcal nuclease, and cytochrome c were consistent with the results of others using orthodox methods and confirm the validity of the method. Where unfolding occurred, it was generally rapidly reversible and the curves were entirely consistent with the presence of only the native and the fully unfolded states. Serum albumin gave more complex curves and a remarkable illustration of micro-heterogeneity. β-Lactoglobulins A and B and ovalbumin refold very slowly and the unfolded molecules appeared to equilibrate preferentially with compact, but non-native, forms at low urea concentrations.     

Interactions of myoglobin with urea and some alkylureas. I. Solvation in urea and alkylurea solutions

The interactions of myoglobin with urea, methyl-, N,N'-dimethyl- and ethylurea in aqueous solutions were studied by density measurements. From the densities at constant chemical potential and constant molality, the partial specific volumes of myoglobin in these solutions as well as the extent of preferential binding of urea and alkylurea to myoglobin were determined. It has been found that water and not the denaturant is preferentially bound in urea solutions and alkylurea solutions up to 4 M so that the Gibbs free energy of myoglobin, i.e., its chemical potential in a denaturant solution, is larger than in water. This behavior of myoglobin is different from that of other globular proteins for which preferential binding of urea has been found. It appears that preferential hydration of myoglobin is due to its high content of ionic groups.     

尿素和包膜尿素对大棚内有害气体浓度变化的影响

通过室内模拟和塑料大棚试验,研究了普通尿素和矿物改性包膜尿素对土壤pH值及大棚内有害气体浓度变化的影响.结果表明,在室内模拟试验条件下,两种氮肥施用初期均导致土壤pH值上升,并于1周后达到最大值,上升幅度超过50％,随后开始下降,至第5周回到初始水平.大棚内施用两种氮肥均使棚内NH₃、NO₂和O₃浓度增加,其中施用普通尿素处理的NH₃、NO₂日均挥发量均大于矿物改性包膜尿素;施用普通尿素处理使大棚内土壤的NH₃、棚内NO₂和O₃的最高浓度达到42.36、41.95和86.00 μg·m^-3·d^-1,3种气体浓度均达到了有害气体伤害植物的临界阈值;NH₃、NO₂挥发强度受棚温和光照强度的影响,O₃浓度随光照强度变化而改变.     

Constraints and missing reactions in the urea cycle.

The stoichiometric relations in a series of biochemical reactions are summarized by a stoichiometric number matrix (with a column for each reaction) and a conservation matrix (with a row for each constraint). These two matrices for a series or cycle of biochemical reactions are related because the columns of the stoichiometric number matrix are in the null space of the conservation matrix, and the rows of the transpose of the conservation matrix are in the null space of the transpose of the stoichiometric number matrix. The conservation matrix for a system of biochemical reactions is of interest because it shows the nature of the constraints in addition to the conservation of atoms and groups. Constraints beyond those for the conservation of atoms and groups indicate "missing reactions" that do not occur because the enzymes involved couple reactions that could occur and still conserve atoms and groups. The interpretation of conservation matrices and stoichiometric matrices for a reaction system is complicated by the fact that they are not unique. However, their row-reduced forms are unique, as are their dimensions, which represent the number of reactants and number of independent reactions. Two matrices that look different contain the same information if they have the same row-reduced form. The urea cycle, which involves five enzyme-catalyzed reactions, and its net reaction are discussed in terms of the linear constraints produced by enzyme catalysis. A procedure to obtain a set of conservation equations that will yield the correct net reaction is described.     

Facilitated transport of urea across the gall-bladder luminal membrane.

Counterflow experiments demonstrate the existence of urea counter-transport on the epithelium luminal surface. This phenomenon disappears when 10(-4) M phloretin is added to the perfusion fluid. Moreover counterflow experiments made using thiourea as elicitor, demonstrate that the phenomenon is specific for the urea.