Dataset Information

Stabilization of the ADP/metaphosphate intermediate during ATP hydrolysis in pre-power stroke myosin: quantitative anatomy of an enzyme.

ABSTRACT: It has been proposed recently that ATP hydrolysis in ATPase enzymes proceeds via an initial intermediate in which the dissociated ?-phosphate of ATP is bound in the protein as a metaphosphate (P?O3(-)). A combined quantum/classical analysis of this dissociated nucleotide state inside myosin provides a quantitative understanding of how the enzyme stabilizes this unusual metaphosphate. Indeed, in vacuum, the energy of the ADP(3-) · P?O3(-) · Mg(2+) complex is much higher than that of the undissociated ATP(4-). The protein brings it to a surprisingly low value. Energy decomposition reveals how much each interaction in the protein stabilizes the metaphosphate state; backbone peptides of the P-loop contribute 50% of the stabilization energy, and the side chain of Lys-185(+) contributes 25%. This can be explained by the fact that these groups make strong favorable interactions with the ?- and ?-phosphates, thus favoring the charge distribution of the metaphosphate state over that of the ATP state. Further stabilization (16%) is achieved by a hydrogen bond between the backbone C=O of Ser-237 (on loop Switch-1) and a water molecule perfectly positioned to attack the P?O3(-) in the subsequent hydrolysis step. The planar and singly negative P?O3(-) is a much better target for the subsequent nucleophilic attack by a negatively charged OH(-) than the tetrahedral and doubly negative P?O4(2-) group of ATP. Therefore, we argue that the present mechanism of metaphosphate stabilization is common to the large family of nucleotide-hydrolyzing enzymes. Methodologically, this work presents a computational approach that allows us to obtain a truly quantitative conception of enzymatic strategy.

SUBMITTER: Kiani FA

PROVIDER: S-EPMC3853302 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Publications

Stabilization of the ADP/metaphosphate intermediate during ATP hydrolysis in pre-power stroke myosin: quantitative anatomy of an enzyme.

Kiani Farooq Ahmad FA Fischer Stefan S

The Journal of biological chemistry 20131028 49

It has been proposed recently that ATP hydrolysis in ATPase enzymes proceeds via an initial intermediate in which the dissociated γ-phosphate of ATP is bound in the protein as a metaphosphate (PγO3(-)). A combined quantum/classical analysis of this dissociated nucleotide state inside myosin provides a quantitative understanding of how the enzyme stabilizes this unusual metaphosphate. Indeed, in vacuum, the energy of the ADP(3-) · PγO3(-) · Mg(2+) complex is much higher than that of the undissoci ...[more]

PMID: 24165121

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Project description:Conventional myosin is representative of biomolecular motors in which the hydrolysis of adenosine triphosphate (ATP) is coupled to large-scale structural transitions both in and remote from the active site. The mechanism that underlies such "mechanochemical coupling," especially the causal relationship between hydrolysis and allosteric structural changes, has remained elusive despite extensive experimental and computational analyses. In this study, using combined quantum mechanical and molecular mechanical simulations and different conformations of the myosin motor domain, we provide evidence to support that regulation of ATP hydrolysis activity is not limited to residues in the immediate environment of the phosphate. Specifically, we illustrate that efficient hydrolysis of ATP depends not only on the proper orientation of the lytic water but also on the structural stability of several nearby residues, especially the Arg238-Glu459 salt bridge (the numbering of residues follows myosin II in Dictyostelium discoideum) and the water molecule that spans this salt bridge and the lytic water. More importantly, by comparing the hydrolysis activities in two motor conformations with very similar active-site (i.e., Switches I and II) configurations, which distinguished this work from our previous study, the results clearly indicate that the ability of these residues to perform crucial electrostatic stabilization relies on the configuration of residues in the nearby N-terminus of the relay helix and the "wedge loop." Without the structural support from those motifs, residues in a closed active site in the post-rigor motor domain undergo subtle structural variations that lead to consistently higher calculated ATP hydrolysis barriers than in the pre-powerstroke state. In other words, starting from the post-rigor state, turning on the ATPase activity requires not only displacement of Switch II to close the active site but also structural transitions in the N-terminus of the relay helix and the "wedge loop," which have been proposed previously to be ultimately coupled to the rotation of the converter subdomain 40 A away.

Project description:Prostate cancer is among the major malignancies that affect men around the world. Adenine nucleotides are important signaling molecules that mediate innumerous biological functions in pathophysiological conditions, including cancer. These molecules are degraded by several ectoenzymes named ectonucleotidases that produce adenosine in the extracellular medium. Some of these ecto-enzymes can be found in soluble in the blood stream. Thus, the present study aimed to evaluate the hydrolysis of adenine nucleotides (ATP, ADP, and AMP) in the plasma blood of patients with prostate cancer. Peripheral blood samples were collected, and questionnaires were filled based on the clinical data of the medical records. The nucleotide hydrolysis was performed by Malachite Green method using ATP, ADP, and AMP as substrates. Plasma from prostate cancer patients presented an elevated hydrolysis of all nucleotides evaluated when compared to healthy individuals. NTPDase inhibitor (ARL67156) and the alkaline phosphatase inhibitor (levamisole) did not alter ATP hydrolysis. However, AMP hydrolysis was reduced by the CD73 inhibitor, APCP, and by levamisole, suggesting the action of a soluble form of CD73 and alkaline phosphatase. On microvesicles, it was observed that there was a low expression and activity of CD39 and almost absent of CD73. The correlation of ATP, ADP, and AMP hydrolysis with clinic pathological data demonstrated that patients who received radiotherapy showed a higher AMP hydrolysis than those who did not, and patients with lower clinical stage (CS-IIA) presented an elevated ATP hydrolysis when compared to those with more advanced clinical stages (CS-IIB and CS-III). Patients of all clinical stages presented an elevated AMPase activity. Therefore, we can suggest that the nucleotide hydrolysis might be attributed to soluble ecto-enzymes present in the plasma, which, in a coordinate manner, produce adenosine in the blood stream, favoring prostate cancer progression.

Dataset Information

Stabilization of the ADP/metaphosphate intermediate during ATP hydrolysis in pre-power stroke myosin: quantitative anatomy of an enzyme.

Publications

Stabilization of the ADP/metaphosphate intermediate during ATP hydrolysis in pre-power stroke myosin: quantitative anatomy of an enzyme.

Similar Datasets

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