Catalyzed Decomposition of Urea. Molecular Dynamics Simulations of the Binding of Urea to Urease

Abstract

We present the results of molecular dynamics simulations on the urea/urease system. The starting structure was prepared from the 2.0Å crystal structure of Benini et al. of DAP-inhibited urease (PDB code 3UBP), 1 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. 2 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 a NH 2 moiety of urea, assisted by Asp360 in the active site. The impact of the simulation results on our understanding of urease catalysis are discussed in detail.The reliable prediction of the mechanism involved in the selectivity and efficiency of enzyme catalyzed reactions continues to challenge both experimental and computational researchers. 3-9 Mutagenic and biochemical procedures, as well as a variety of computational methodologies have been applied with the goal of understanding the enzymatic effect on reactivity, and several working hypotheses have been proposed. Among them, there is general agreement of the role played by the reduction of the free energy of activation, 3 but the origins of the free energy lowering, which involves non-covalent and covalent effects, is the subject of ongoing debate. 10 Non-covalent factors involve transition state electrostatic stabilization, 11 ground state destabilization and desolvation, 12 reduction of reorganization energy by binding in near attack conformations, 13 entropy trapping, 14 as well as several other related effects. Covalent and non-covalent effects are not exclusive, and rate enhancement can come from both effects, as well as from others. Covalent factors are dominant, whenever present. 15 They usually occur in metalloenzymes, where the metal centers covalently bind the substrate, stabilizing the transition state complex and leading to highly proficient catalysts. 10, 16 This is the case of the metalloenzyme urease, whose proficiency is presently a matter of extensive discussion, as values of 10 17 and 10 32 have been determined by experimental and theoretical means. 17, 18Urea amidohydrolase (urease) is a nickel-containing enzyme that catalyses the hydrolysis of urea to produce ammonia and carbon dioxide in the last step of nitrogen mineralization. 1, 19-25 The mechanism of the catalyzed reaction continues to be of interest, and there is still no agreement on whether the reaction involves a carbamic acid intermediate, a cyanate * To whom correspondence should be addressed. .edu † Present address: Department of Chemistry, Quantum Theory Project, ...

Interplay of metal ions and urease

“…Computational analysis of the bdptz complex mentioned above led to the suggestion that urea may decompose by both hydrolytic and elimination routes 41. Molecular dynamics simulations were used to both assess the likely protonation status of His 219 and His 320 and compare various urea decomposition pathways 42. More recent quantum chemical studies extend the examination of various pathways and come to the same conclusion as above that hydrolysis and elimination reactions may compete in the enzyme 43.…”

Biosynthesis of the Urease Metallocenter

“…1D), with two Ni 2ϩ ions bridged by a carbamylated Lys residue and water; one metal additionally coordinates two His residues and a terminal water molecule, and the second Ni 2ϩ ion also coordinates two His residues, one Asp residue, and water. Aspects of the enzyme mechanism remain controversial (7,9,(15)(16)(17), but most proposals suggest that the urea carbonyl oxygen displaces the terminal water from the Ni 2ϩ ion shown on the left (Fig. 1D), with another Ni 2ϩ -bound water molecule acting as a nucleophile to achieve catalysis.…”

Experimental versus theoretical evidence for the rate‐limiting steps in uncatalyzed and H+‐ and HO−‐catalyzed hydrolysis of the amide bond

“…The urease‐catalyzed cleavage of urea has been analyzed by means of modern theoretical methodologies, which have revealed the free energy of activation closed to 30 kcal mol −1 , i.e., the first‐order rate constant of the order of 10 −10 s −1 at 25°C 53,58. The results obtained from the recent molecular dynamic simulations conclude a possible competition between bimolecular Ad–El and elimination mechanisms 53,59. The experimentally determined value of k 0 (6.5 × 10 −10 M −1 s −1 at 25°C) for the cleavage of urea by the elimination mechanism 41 is approximately 54‐fold larger than the estimated value of k 0 (1.2 × 10 −11 M −1 s −1 at 25°C) for the cleavage of urea through the Ad–El mechanism 40.…”