Theoretical Study of the Phosphoryl Transfer Reaction from ATP to Dha Catalyzed by DhaK from Escherichia coli

Abstract

Protein kinases, representing one of the largest protein family involved in almost all aspects of cell life, have become one of the most important targets for the development of new drugs to be used in, for instance, cancer treatments. In this paper an exhaustive theoretical study of the phosphoryl transfer reaction from adenosine triphosphate (ATP) to dihydroxyacetone (Dha) catalyzed by DhaK from Escherichia coli (E. coli) is reported. Two different mechanisms, previously proposed for the phosphoryl transfer from ATP to the hydroxyl side chain of specific serine, threonine or tyrosine residues, have been explored based on the generation of free energy surfaces (FES) computed with hybrid QM/MM potentials. The results suggest that the substrate-assisted phosphoryl and proton-transfer mechanism is kinetically more favorable than the mechanism where an aspartate would be activating the Dha. Although the details of the mechanisms appear to be dramatically dependent on the level of theory employed in the calculations (PM3/MM, B3LYP:PM3/MM or B3LYP/MM), the transition states (TSs) for the phosphoryl transfer step appear to be described as a concerted step with different degrees of synchronicity in the breaking and forming bonds process in both explored mechanisms. Residues of the active site belonging to different subunits of the protein such as Gly78B, Thr79A, Ser80A, Arg178B and one Mg 2+ cation would be stabilizing the transferred phosphate in the TS. Asp109A would have a structural role by posing the Dha and other residues of the active site in the proper orientation. The information derived from our calculations not only reveals the role of the enzyme and the particular residues of its active site, but it can assist in the rationally design of new more specific inhibitors.3

Computational study of the phosphoryl donor activity of dihydroxyacetone kinase from ATP to inorganic polyphosphate

“…These values are significantly lower than those reported for the asp‐assisted mechanism in the wild‐type and in the mutated enzyme. This trend is in agreement with our previous study of the phosphate transfer from ATP to Dha catalyzed by DHAK that also showed the substrate‐assisted mechanism as the most favorable one …”

“…In the substrate‐assisted mechanism the poly‐P molecule directly abstracts the proton from Dha. De Vivo et al supported this mechanism in the theoretical study of the phosphate transfer reaction from ATP to a serine residue in CDK2, as well as our previous study of the phosphate transfer reaction from ATP to Dha on wild‐type DHAK . Both reaction mechanisms are explored for the phosphate transfer reaction from poly‐P, instead of ATP, to Dha on wild‐type and E526K mutant in the present study.…”

“…Lower energy TS conformations could be localized within this protocol thus providing much lower energy barriers. Moreover, as already observed in our previous study of the phosphoryl transfer reaction from ATP to Dha catalyzed by DhaK from E. coli , there must be a lack of overlapping between the B3LYP and PM3 conformational space. Then, since the starting structures used to locate and optimize the B3LYP/MM stationary point structures were those derived from the PM3/MM calculations, they are not necessary the lowest energy ones, especially for the TSs structures.…”

“…Consequently, it has been employed widely to model phosphorus and phosphate groups successfully . In fact, the semiempirical Hamiltonian PM3 was already used in our group to describe the QM subset of atoms in our previous QM/MM studies of the phosphate transfer reaction between ATP and Dha in aqueous solution and catalysed by DHAK . In addition, the B3LYP functional, with the 6–31G(d,p) basis set, was also used to treat the QM region of the system combining fDYNAMO library with Gaussian09 program .…”

“…However, De Vivo et al, based on theoretical results derived from gas‐phase DFT calculations, classical molecular dynamics (MD), and quantum mechanics/molecular mechanics (QM/MM) Car–Parrinello simulations, suggested a substrate‐assisted mechanism for the phosphorylation reaction in cyclin‐dependent kinase (CDK2) where ATP was shown to take the proton from a serine residue . We have recently studied the phosphoryl transfer mechanism from ATP to Dha in DHAK from E. coli finding the substrate‐assisted mechanism kinetically more favorable than the asp‐assisted mechanism …”