Associative mechanism for phosphoryl transfer: A molecular dynamics simulation of Escherichia coli adenylate kinase complexed with its substrates

Abstract

The ternary complex of Escherichia coli adenylate kinase (ECAK) with its substrates adenosine monophosphate (AMP) and Mg-ATP, which catalyzes the reversible transfer of a phosphoryl group between adenosine triphosphate (ATP) and AMP, was studied using molecular dynamics. The starting structure for the simulation was assembled from the crystal structures of ECAK complexed with the bisubstrate analog diadenosine pentaphosphate (AP(5)A) and of Bacillus stearothermophilus adenylate kinase complexed with AP(5)A, Mg(2+), and 4 coordinated water molecules, and by deleting 1 phosphate group from AP(5)A. The interactions of ECAK residues with the various moieties of ATP and AMP were compared to those inferred from NMR, X-ray crystallography, site-directed mutagenesis, and enzyme kinetic studies. The simulation supports the hypothesis that hydrogen bonds between AMP's adenine and the protein are at the origin of the high nucleoside monophosphate (NMP) specificity of AK. The ATP adenine and ribose moieties are only loosely bound to the protein, while the ATP phosphates are strongly bound to surrounding residues. The coordination sphere of Mg(2+), consisting of 4 waters and oxygens of the ATP beta- and gamma-phosphates, stays approximately octahedral during the simulation. The important role of the conserved Lys13 in the P loop in stabilizing the active site by bridging the ATP and AMP phosphates is evident. The influence of Mg(2+), of its coordination waters, and of surrounding charged residues in maintaining the geometry and distances of the AMP alpha-phosphate and ATP beta- and gamma-phosphates is sufficient to support an associative reaction mechanism for phosphoryl transfer.

hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: Structural and functional studies

“…Approach of a mobile AMP towards an immobile ATP is consistent with a transfer mechanism in which the AMP -phosphate attacks thephosphate of ATP, assisted by water molecules that bridge ATP -and AMPphosphates. 49 Our predicted IFD model geometries and interphosphate (ATP-ADP) P-P distances are in agreement with MD predictions for the homologous E. coli AK-Mg −1-ATP-ADP complex. 49 Despite this, our above successful test-validation of the IFD method and the documented successes of IFD in many other recent applications [50][51][52][53][54][55] docking models obviously have to be treated with a reasonable measure of caution, like any prediction approaches.…”

“…49 Our predicted IFD model geometries and interphosphate (ATP-ADP) P-P distances are in agreement with MD predictions for the homologous E. coli AK-Mg −1-ATP-ADP complex. 49 Despite this, our above successful test-validation of the IFD method and the documented successes of IFD in many other recent applications [50][51][52][53][54][55] docking models obviously have to be treated with a reasonable measure of caution, like any prediction approaches. Finally, also of note in this context is a recent publication by Nucci et al, 56 which highlights the limitations of the "static" picture of water molecule positions, as given by crystal structure determination, compared with a more detailed dynamic picture, which can be produced by NMR reverse micelle technology.…”

“…1 and 2(A)]. In addition, the higher-resolution structure allowed us to build accurately the nucleoside monophosphate-binding loop (NMP-binding loop; amino acids [45][46][47][48][49][50][51][52][53][54][55], whose residues were interpreted as disordered in a previous structural analysis of hCINAP. 2 In our structure (Fig.…”

Conformational Transitions of Adenylate Kinase: Switching by Cracking

“…B-factors for all simulations were significantly larger than crystal structure B-factors, which is expected when comparing simulated to crystallographic B-factors [32]. Large B-factors have also been observed in all-atom, explicit solvent, simulations and were attributed to not including crystal contacts [33]. To validate this claim, we simulated the dimeric form (PDB entry 4AKE:chains A and B) of the open conformation using a structure-based potential.…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…Mg 2ϩ , which is necessary for adenylate kinase activity, does not coordinate to side chains of the enzyme but rather coordinates to two oxygens of di(adenosine-5=)pentaphosphate as well as to four water molecules (206). This binding of Mg 2ϩ to di(adenosine-5=)pentaphosphate is consistent with ␤,␥-MgATP as the substrate for adenylate kinase (207) and Mg 2ϩ coordination to oxygens of both of the ␤-phosphates of two ADP molecules of M. tuberculosis adenylate kinase, i.e., the reaction in the direction of ATP synthesis (PDB code 2cdn) (208). Furthermore, in comparison with diphosphoryl and nucleotidyl transfer reactions described above, adenylate kinase may represent an alternative means of performing the function played by the second Mg 2ϩ in other phosphotransferases.…”