Insights from Affinity Labeling into Nucleotide- and Coenzyme-Dependent Enzyme Catalysis and Regulation

Colman, Roberta F.

doi:10.2174/157018006778194853

Cited by 3 publications

(1 citation statement)

References 52 publications

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“…Affinity labeling is a powerful technique for identifying the amino acids which comprise the substrate or coenzyme binding sites of enzymes (as recently reviewed [1]). Although these sites can be inferred from the three-dimensional structure determined by X-ray or NMR analysis, less than 20% of human proteins have had such structures determined.…”

Section: Introductionmentioning

confidence: 99%

A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

Hung

Liu

Pixley

et al. 2008

Bioorganic Chemistry

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The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3′, 5′-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (K I = 43.4 ± 7.2 μM and k cart = 0.007 ± 0.0006 min −1 ). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (K d = 72 μM) > Sp-cGMPS (124), Sp-cAMPS (182) > GMP (1517), Rp-cAMPS (3762), AMP (4370 μM). NAD + , neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes.

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Section: Introductionmentioning

confidence: 99%

A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

Hung

Liu

Pixley

et al. 2008

Bioorganic Chemistry

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Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry

Cravatt

Wright

Kozarich³

2008

Annu. Rev. Biochem.

1,132

1,047

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Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chemical proteomic strategy to characterize enzyme function directly in native biological systems on a global scale. Here, we review the basic technology of ABPP, the enzyme classes addressable by this method, and the biological discoveries attributable to its application.

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Synthesis and Evaluation of a Novel Adenosine-Ribose Probe for Global-Scale Profiling of Nucleoside and Nucleotide-Binding Proteins

et al. 2015

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Proteomics is a powerful approach used for investigating the complex molecular mechanisms of disease pathogenesis and progression. An important challenge in modern protein profiling approaches involves targeting of specific protein activities in order to identify altered molecular processes associated with disease pathophysiology. Adenosine-binding proteins represent an important subset of the proteome where aberrant expression or activity changes of these proteins have been implicated in numerous human diseases. Herein, we describe an affinity-based approach for the enrichment of adenosine-binding proteins from a complex cell proteome. A novel N 6-biotinylated-8-azido-adenosine probe (AdoR probe) was synthesized, which contains a reactive group that forms a covalent bond with the target proteins, as well as a biotin tag for affinity enrichment using avidin chromatography. Probe specificity was confirmed with protein standards prior to further evaluation in a complex protein mixture consisting of a lysate derived from mouse neuroblastoma N18TG2 cells. Protein identification and relative quantitation using mass spectrometry allowed for the identification of small variations in abundance of nucleoside- and nucleotide-binding proteins in these samples where a significant enrichment of AdoR-binding proteins in the labeled proteome from the neuroblastoma cells was observed. The results from this study demonstrate the utility of this method to enrich for nucleoside- and nucleotide-binding proteins in a complex protein mixture, pointing towards a unique set of proteins that can be examined in the context of further understanding mechanisms of disease, or fundamental biological processes in general.

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Insights from Affinity Labeling into Nucleotide- and Coenzyme-Dependent Enzyme Catalysis and Regulation

Cited by 3 publications

References 52 publications

A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3′,5′-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry

Synthesis and Evaluation of a Novel Adenosine-Ribose Probe for Global-Scale Profiling of Nucleoside and Nucleotide-Binding Proteins

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