Enzymatic reactions


06/02/2008, Trieste, SISSA


Empirical valence bond simulations of the chemical mechanism of ATP to cAMP conversion by anthrax edema factor
L Mones, WJ Tang and J Florian
2013, Biochemistry 52 (15), 2672-2682
Abstract: The two-metal catalysis by the adenylyl cyclase domain of the anthrax edema factor toxin was simulated using the empirical valence bond (EVB) quantum mechanical/molecular mechanical approach. These calculations considered the energetics of the nucleophile deprotonation and the formation of a new P–O bond in aqueous solution and in the enzyme–substrate complex present in the crystal structure models of the reactant and product states of the reaction. Our calculations support a reaction pathway that involves metal-assisted transfer of a proton from the nucleophile to the bulk aqueous solution followed by subsequent formation of an unstable pentavalent intermediate that decomposes into cAMP and pyrophosphate (PPi). This pathway involves ligand exchange in the first solvation sphere of the catalytic metal. At 12.9 kcal/mol, the barrier for the last step of the reaction, the cleavage of the P–O bond to PPi, corresponds to the highest point on the free energy profile for this reaction pathway. However, this energy is too close to the value of 11.4 kcal/mol calculated for the barrier of the nucleophilic attack step to reach a definitive conclusion about the rate-limiting step. The calculated reaction mechanism is supported by reasonable agreement between the experimental and calculated catalytic rate constant decrease caused by the mutation of the active site lysine 346 to arginine.

On the divalent metal ion dependence of DNA cleavage by restriction endonucleases of the EcoRI family
V Pingoud, W Wende, P Friedhoff, M Reuter, J Alves, A Jeltsch, L Mones, M Fuxreiter and A Pingoud
2009, Journal of molecular biology 393 (1), 140-160
Abstract: Restriction endonucleases of the PD…D/EXK family need Mg2+ for DNA cleavage. Whereas Mg2+ (or Mn2+) promotes catalysis, Ca2+ (without Mg2+) only supports DNA binding. The role of Mg2+ in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg2+ involved in catalysis. To address this problem, we measured the Mg2+ and Mn2+ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me2+ per active site. DNA cleavage experiments were carried out at various Mg2+ and Mn2+ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg2+ and Mn2+ concentration dependence. In general, the Mg2+ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn2+ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg2+ or Mn2+ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca2+. Based on these results, we propose that one Mg2+ or Mn2+ is critical for restriction enzyme activation, and binding of a second Me2+ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg2+ or Mn2+ mainly leads to an increase in Km, such that the inhibitory effect of excess Mg2+ or Mn2+ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me2+ binding to these enzymes.

Probing the Two-Metal Ion Mechanism in the Restriction Endonuclease BamHI
L Mones, P Kulhánek, J Florian, I Simon and M Fuxreiter
2007, Biochemistry 46 (50), 14514-14523
Abstract: The choreography of restriction endonuclease catalysis is a long-standing paradigm in molecular biology. Bivalent metal ions are required almost for all PD..D/ExK type enzymes, but the number of cofactors essential for the DNA backbone scission remained ambiguous. On the basis of crystal structures and biochemical data for various restriction enzymes, three models have been developed that assign critical roles for one, two, or three metal ions during the phosphodiester hydrolysis. To resolve this apparent controversy, we investigated the mechanism of BamHI catalysis using quantum mechanical/molecular mechanical simulation techniques and determined the activation barriers of three possible pathways that involve a Glu-113 or a neighboring water molecule as a general base or an external nucleophile that penetrated from bulk solution. The extrinsic mechanism was found to be the most favorable with an activation free energy of 23.4 kcal/mol, in reasonable agreement with the experimental data. On the basis of the effect of the individual metal ions on the activation barrier, metal ion A was concluded to be pivotal for the reaction, while the enzyme lacking metal ion B still has moderate efficiency. Thus, we propose that the catalytic scheme of BamHI does not involve a general base for nucleophile generation and requires one obligatory metal ion for catalysis that stabilizes the attacking nucleophile and coordinates it throughout the nucleophilic attack. Such a model may also explain the variation in the number of metal ions in the crystal structures and thus could serve as a framework for a unified catalytic scheme of type II restriction endonucleases.

Metal-binding sites at the active site of restriction endonuclease BamHI can conform to a one-ion mechanism
L Mones, I Simon and M Fuxreiter
2007, Biological Chemistry 388 (1), 73-78
Abstract: The number of metal ions required for phosphoryl transfer in restriction endonucleases is still an unresolved question in molecular biology. The two Ca2+ and Mn2+ ions observed in the pre- and post-reactive complexes of BamHI conform to the classical two-metal ion choreography. We probed the Mg2+ cofactor positions at the active site of BamHI by molecular dynamics simulations with one and two metal ions present and identified several catalytically relevant sites. These can mark the pathway of a single ion during catalysis, suggesting its critical role, while a regulatory function is proposed for a possible second ion.