Anthrax Edema Factor: An Ion-Adaptive Mechanism of Catalysis with Increased Transition-State Conformational Flexibility

Abstract

Edema Factor (EF) is one of three major toxins of anthrax. EF is an adenylyl cyclase that disrupts cell signaling by accelerating the conversion of ATP into cyclic-AMP. EF has a much higher catalytic rate than that of mammalian adenylyl cyclases (mACs). Crystal structures were obtained for mACs and EF, but the molecular basis for different catalytic activities remained poorly understood. In particular, the arrangement of the active site in EF is unclear in what concerns the number of ions present and the conformation of the substrate. Here, we use quantum mechanics-molecular mechanics simulations to estimate the free-energy profiles for the reaction catalyzed by EF and a mAC. We found that EF catalysis is possible, and faster than that of mACs, in both one and two Mg(2+)-ion-binding modes, providing adaptive plasticity to host-cell environments. In both enzymes, the reaction mechanisms are highly associative. However, mechanistic differences exist. In the mAC, the nucleophile oxygen (ATP-O3') is consistently coordinated to one of the Mg(2+) ions, increasing its acidity. In EF, on the other hand, this coordination is eventual and not essential for the reaction to proceed. The persistent coordination of O3' to the ion is favored in mACs by a greater ion partial charge. In EF, the reduced acidity of the O3' oxygen is compensated by the presence of the His351 residue for proton abstraction. As proton transfer in EF does not require persistent attachment of the substrate to an ion, the substrate (ATP) and transition state display greater conformational flexibilities. These greater flexibilities allow the sampling of lower-energy conformations and might represent an entropic advantage for catalytic efficiency.

Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires

“…Jara and Martínez 5 describe a single-step mechanism with strongly overestimated energy barriers and without specification of the proton transfer pathway. Both studies 4,5 agree that this reaction refers to the mechanism with a penta-coordinated structure of the Tentative reaction mechanisms are also discussed in experimental studies. Liu et al 11 notice that a conservative Asp residue stands in an optimal position to the ribose 3' hydroxyl to serve as a catalytic base.…”

“…S1). These points refer to The latter serves either as a final proton acceptor 3,5 or as a proton shuttle in an appropriate proton wire route. 4 Table 1.…”

“…1 It is enough to mention a role of adenylyl cyclases in human health and diseases due to key regulatory functions of these enzymes in essentially all cells. 2 Numerous biochemical, biological and biomedical papers discuss various aspects of structure and function of ACs, some of them tentatively formulate catalytic mechanisms of the reaction ATP → cAMP inside enzyme active sites, and only three papers [3][4][5] report somewhat inconsistent results of quantum-based simulations of this reaction in proteins. Fig.…”

Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires

“…We pay attention to the following structural elements in these candidates for a productive ES complex: (i) the O 3' -P A distance, (ii) orientation of the The latter serves either as a final proton acceptor 3,5 or as a proton shuttle in an appropriate proton wire route. 4 Structures ES B and ES C lying about 5 and 10 kcal/mol higher in energy than ES A exhibit a different orientation of the O 3' H 3' group.…”

“…6 Experimentally, the protein was crystallized with an ATP analog, adenosine 5′-(α-thio)-triphosphate (ATPαS-Rp), one magnesium ion Mg A 2+ and one manganese atom Mn B 2+ . 6 The authors of the computational paper 5 created model systems restoring proteins with ATP and two magnesium ions in the active sites, carried out molecular dynamics (MD) simulations with the classical and QM/MM potentials. The semi-empirical density functional tight-binding method with the self-consistent charge approach (SCC-DFTB) 8,9 was applied in QM/MM simulations.…”