Mapping Enzyme Active Sites in Complex Proteomes.
Saved in:
| Title: | Mapping Enzyme Active Sites in Complex Proteomes. |
|---|---|
| Authors: | Adam, Gregory C.1, Burbaum, Jonathan2, Kozarich, John W.2, Patricelli, Matthew P.2 mattp@activx.com, Cravatt, Benjamin F.1 cravatt@scripps.edu |
| Source: | Journal of the American Chemical Society. 2/11/2004, Vol. 126 Issue 5, p1363-1368. 6p. |
| Subjects: | Genomes, Enzymes, Aldehyde dehydrogenase, Prokaryotes, Glutathione, Gel electrophoresis, Isotopes |
| Abstract: | Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel- free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3βhydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites. [ABSTRACT FROM AUTHOR] |
| Copyright of Journal of the American Chemical Society is the property of American Chemical Society and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
| FullText | Text: Availability: 0 |
|---|---|
| Header | DbId: egs DbLabel: Engineering Source An: 12420957 AccessLevel: 6 PubType: Academic Journal PubTypeId: academicJournal PreciseRelevancyScore: 0 |
| IllustrationInfo | |
| Items | – Name: Title Label: Title Group: Ti Data: Mapping Enzyme Active Sites in Complex Proteomes. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Adam%2C+Gregory+C%2E%22">Adam, Gregory C.</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Burbaum%2C+Jonathan%22">Burbaum, Jonathan</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Kozarich%2C+John+W%2E%22">Kozarich, John W.</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Patricelli%2C+Matthew+P%2E%22">Patricelli, Matthew P.</searchLink><relatesTo>2</relatesTo><i> mattp@activx.com</i><br /><searchLink fieldCode="AR" term="%22Cravatt%2C+Benjamin+F%2E%22">Cravatt, Benjamin F.</searchLink><relatesTo>1</relatesTo><i> cravatt@scripps.edu</i> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22Journal+of+the+American+Chemical+Society%22">Journal of the American Chemical Society</searchLink>. 2/11/2004, Vol. 126 Issue 5, p1363-1368. 6p. – Name: Subject Label: Subjects Group: Su Data: <searchLink fieldCode="DE" term="%22Genomes%22">Genomes</searchLink><br /><searchLink fieldCode="DE" term="%22Enzymes%22">Enzymes</searchLink><br /><searchLink fieldCode="DE" term="%22Aldehyde+dehydrogenase%22">Aldehyde dehydrogenase</searchLink><br /><searchLink fieldCode="DE" term="%22Prokaryotes%22">Prokaryotes</searchLink><br /><searchLink fieldCode="DE" term="%22Glutathione%22">Glutathione</searchLink><br /><searchLink fieldCode="DE" term="%22Gel+electrophoresis%22">Gel electrophoresis</searchLink><br /><searchLink fieldCode="DE" term="%22Isotopes%22">Isotopes</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel- free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3βhydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of Journal of the American Chemical Society is the property of American Chemical Society and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
| PLink | https://search.ebscohost.com/login.aspx?direct=true&site=eds-live&db=egs&AN=12420957 |
| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1021/ja038441g Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 6 StartPage: 1363 Subjects: – SubjectFull: Genomes Type: general – SubjectFull: Enzymes Type: general – SubjectFull: Aldehyde dehydrogenase Type: general – SubjectFull: Prokaryotes Type: general – SubjectFull: Glutathione Type: general – SubjectFull: Gel electrophoresis Type: general – SubjectFull: Isotopes Type: general Titles: – TitleFull: Mapping Enzyme Active Sites in Complex Proteomes. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Adam, Gregory C. – PersonEntity: Name: NameFull: Burbaum, Jonathan – PersonEntity: Name: NameFull: Kozarich, John W. – PersonEntity: Name: NameFull: Patricelli, Matthew P. – PersonEntity: Name: NameFull: Cravatt, Benjamin F. IsPartOfRelationships: – BibEntity: Dates: – D: 11 M: 02 Text: 2/11/2004 Type: published Y: 2004 Identifiers: – Type: issn-print Value: 00027863 Numbering: – Type: volume Value: 126 – Type: issue Value: 5 Titles: – TitleFull: Journal of the American Chemical Society Type: main |
| ResultId | 1 |
