Dataset Information

Price to be paid for two-metal catalysis: magnesium ions that accelerate chemistry unavoidably limit product release from a protein kinase.

ABSTRACT: Incorporation of divalent metal ions into an active site is a fundamental catalytic tool used by diverse enzymes. Divalent cations are used by protein kinases to both stabilize ATP binding and accelerate chemistry. Kinetic analysis establishes that Cyclin-dependent kinase 2 (CDK2) requires simultaneous binding of two Mg(2+) ions for catalysis of phosphoryl transfer. This tool, however, comes with a price: the rate-acceleration effects are opposed by an unavoidable rate-limiting consequence of the use of two Mg(2+) ions by CDK2. The essential metal ions stabilize ADP product binding and limit the overall rate of the reaction. We demonstrate that product release is rate limiting for activated CDK2 and evaluate the effects of the two catalytically essential Mg(2+) ions on the stability of the ADP product within the active site. We present two new crystal structures of CDK2 bound to ADP showing how the phosphate groups can be coordinated by either one or two Mg(2+) ions, with the occupancy of one site in a weaker equilibrium. Molecular dynamics simulations indicate that ADP phosphate mobility is more restricted when ADP is coordinated by two Mg(2+) ions compared to one. The structural similarity between the rigid ADP·2Mg product and the cooperatively assembled transition state provides a mechanistic rational for the rate-limiting ADP release that is observed. We demonstrate that although the simultaneous binding of two Mg(2+) ions is essential for efficient phosphoryl transfer, the presence of both Mg(2+) ions in the active site also cooperatively increases ADP affinity and opposes its release. Evolution of protein kinases must have involved careful tuning of the affinity for the second Mg(2+) ion in order to balance the needs to stabilize the chemical transition state and allow timely product release. The link between Mg(2+) site affinity and activity presents a chemical handle that may be used by regulatory factors as well as explain some mutational effects.

SUBMITTER: Jacobsen DM

PROVIDER: S-EPMC3446636 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Price to be paid for two-metal catalysis: magnesium ions that accelerate chemistry unavoidably limit product release from a protein kinase.

Jacobsen Douglas M DM Bao Zhao-Qin ZQ O'Brien Patrick P Brooks Charles L CL Young Matthew A MA

Journal of the American Chemical Society 20120907 37

Incorporation of divalent metal ions into an active site is a fundamental catalytic tool used by diverse enzymes. Divalent cations are used by protein kinases to both stabilize ATP binding and accelerate chemistry. Kinetic analysis establishes that Cyclin-dependent kinase 2 (CDK2) requires simultaneous binding of two Mg(2+) ions for catalysis of phosphoryl transfer. This tool, however, comes with a price: the rate-acceleration effects are opposed by an unavoidable rate-limiting consequence of th ...[more]

PMID: 22891849

Similar Datasets

Project description:BackgroundFor enzymes with buried active sites, transporting substrates/products ligands between active sites and bulk solvent via access tunnels is a key step in the catalytic cycle of these enzymes. Thus, tunnel engineering is becoming a powerful strategy to refine the catalytic properties of these enzymes. The tunnel-like structures have been described in enzymes catalyzing bulky substrates like glycosyl hydrolases, while it is still uncertain whether these structures involved in ligands exchange. Till so far, no studies have been reported on the application of tunnel engineering strategy for optimizing properties of enzymes catalyzing biopolymers.ResultsIn this study, xylanase S7-xyl (PDB: 2UWF) with a deep active cleft was chosen as a study model to evaluate the functionalities of tunnel-like structures on the properties of biopolymer-degrading enzymes. Three tunnel-like structures in S7-xyl were identified and simultaneously reshaped through multi-sites saturated mutagenesis; the most advantageous mutant 254RL1 (V207N/Q238S/W241R) exhibited 340% increase in specific activity compared to S7-xyl. Deconvolution analysis revealed that all three mutations contributed synergistically to the improved activity of 254RL1. Enzymatic characterization showed that larger end products were released in 254RL1, while substrate binding and structural stability were not changed. Dissection of the structural alterations revealed that both the tun_1 and tun_2 in 254RL1 have larger bottleneck radius and shorter length than those of S7-xyl, suggesting that these tunnel-like structures may function as products transportation pathways. Attributed to the improved catalytic efficiency, 254RL1 represents a superior accessory enzyme to enhance the hydrolysis efficiency of cellulase towards different pretreated lignocellulose materials. In addition, tunnel engineering strategy was also successfully applied to improve the catalytic activities of three other xylanases including xylanase NG27-xyl from Bacillus sp. strain NG-27, TSAA1-xyl from Geobacillus sp. TSAA1 and N165-xyl from Bacillus sp. N16-5, with 80%, 20% and 170% increase in specific activity, respectively.ConclusionsThis study represents a pilot study of engineering and functional verification of tunnel-like structures in enzymes catalyzing biopolymer. The specific activities of four xylanases with buried active sites were successfully improved by tunnel engineering. It is highly likely that tunnel reshaping can be used to engineer better biomass-degrading abilities in other lignocellulolytic enzymes with buried active sites.

Project description:BackgroundPrior studies have demonstrated how price transparency lowers the test-ordering rates of trainees in hospitals, and physician-targeted price transparency efforts have been viewed as a promising cost-controlling strategy.ObjectiveTo examine the effect of displaying paid-price information on test-ordering rates for common imaging studies and procedures within an accountable care organization (ACO).DesignBlock randomized controlled trial for 1 year.SubjectsA total of 1205 fully licensed clinicians (728 primary care, 477 specialists).InterventionStarting January 2014, clinicians in the Control arm received no price display; those in the intervention arms received Single or Paired Internal/External Median Prices in the test-ordering screen of their electronic health record. Internal prices were the amounts paid by insurers for the ACO's services; external paid prices were the amounts paid by insurers for the same services when delivered by unaffiliated providers.Main measuresOrdering rates (orders per 100 face-to-face encounters with adult patients): overall, designated to be completed internally within the ACO, considered "inappropriate" (e.g., MRI for simple headache), and thought to be "appropriate" (e.g., screening colonoscopy).Key resultsWe found no significant difference in overall ordering rates across the Control, Single Median Price, or Paired Internal/External Median Prices study arms. For every 100 encounters, clinicians in the Control arm ordered 15.0 (SD 31.1) tests, those in the Single Median Price arm ordered 15.0 (SD 16.2) tests, and those in the Paired Prices arms ordered 15.7 (SD 20.5) tests (one-way ANOVA p-value 0.88). There was no difference in ordering rates for tests designated to be completed internally or considered to be inappropriate or appropriate.ConclusionsDisplaying paid-price information did not alter how frequently primary care and specialist clinicians ordered imaging studies and procedures within an ACO. Those with a particular interest in removing waste from the health care system may want to consider a variety of contextual factors that can affect physician-targeted price transparency.

Dataset Information

Price to be paid for two-metal catalysis: magnesium ions that accelerate chemistry unavoidably limit product release from a protein kinase.

Publications

Price to be paid for two-metal catalysis: magnesium ions that accelerate chemistry unavoidably limit product release from a protein kinase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets