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Article: The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries
Title | The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries |
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Authors | |
Issue Date | 1998 |
Publisher | American Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistry |
Citation | Biochemistry, 1998, v. 37 n. 38, p. 13239-13249 How to Cite? |
Abstract | Human recombinant CD38 catalyzes the formation of both cyclic ADP- ribose and ADP-ribose products from NAD+ and hydrolyzes cyclic ADP-ribose to ADP-ribose. The corresponding GDP products are formed from NGD+. The enzyme was characterized by substrate and inhibition kinetics, exchange studies, rapid-quench reactions, and stopped-flow-fluorescence spectroscopy to establish the reaction mechanism and energetics for individual steps. Noncyclizable substrates NMN+ and nicotinamide-7-deaza-hypoxanthine dinucleotide (7-deaza NHD+) were rapidly hydrolyzed by the enzyme. The k(cat) for NMN+ was 5-fold higher than that of NAD+ and has the greatest reported kcat of any substrate for CD38. 7-deaza-NHD+ was hydrolyzed at approximately one-third the rate of NHD+ but does not form a cyclic product. These results establish that a cyclic intermediate is not required for substrate hydrolysis. The ratio of methanolysis to hydrolysis for cADPR and NAD+ catalyzed by CD38 increases linearly with MeOH concentration. Both reactions produce predominantly the β-methoxy riboside compound, with a relative nucleophilicity of MeOH to H2O of 11. These results indicate the existence of a stabilized cationic intermediate for all observed chemistries in the active site of CD38. The partitioning of this intermediate between cyclization, hydrolysis, and nicotinamide-exchange unites the mechanisms of CD38 chemistries. Steady-state and pre-steady-state parameters for the partition and exchange mechanisms allowed full characterization of the reaction coordinate. Stopped-flow methods indicate a burst of cGDPR formation followed by the steady-state reaction rate. A lag phase, which was NGD+ concentration dependent, was also observed. The burst size indicates that the dimeric enzyme has a single catalytic site formed by two subunits. Pre- steady-state quench experiments did not detect covalent intermediates. Nicotinamide hydrolysis of NGD+ precedes cyclization and the chemical quench decomposes the enzyme-bound species to a mixture of cyclic and hydrolysis products. The time dependence of this ratio indicated that nicotinamide bond- breakage occurs 4 times faster than the conversion of the intermediate to products. Product release is the overall rate-limiting step for enzyme reaction with NGD+. |
Persistent Identifier | http://hdl.handle.net/10722/171658 |
ISSN | 2023 Impact Factor: 2.9 2023 SCImago Journal Rankings: 1.042 |
ISI Accession Number ID | |
References |
DC Field | Value | Language |
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dc.contributor.author | Sauve, AA | en_US |
dc.contributor.author | Munshi, C | en_US |
dc.contributor.author | Lee, HC | en_US |
dc.contributor.author | Schramm, VL | en_US |
dc.date.accessioned | 2012-10-30T06:16:12Z | - |
dc.date.available | 2012-10-30T06:16:12Z | - |
dc.date.issued | 1998 | en_US |
dc.identifier.citation | Biochemistry, 1998, v. 37 n. 38, p. 13239-13249 | en_US |
dc.identifier.issn | 0006-2960 | en_US |
dc.identifier.uri | http://hdl.handle.net/10722/171658 | - |
dc.description.abstract | Human recombinant CD38 catalyzes the formation of both cyclic ADP- ribose and ADP-ribose products from NAD+ and hydrolyzes cyclic ADP-ribose to ADP-ribose. The corresponding GDP products are formed from NGD+. The enzyme was characterized by substrate and inhibition kinetics, exchange studies, rapid-quench reactions, and stopped-flow-fluorescence spectroscopy to establish the reaction mechanism and energetics for individual steps. Noncyclizable substrates NMN+ and nicotinamide-7-deaza-hypoxanthine dinucleotide (7-deaza NHD+) were rapidly hydrolyzed by the enzyme. The k(cat) for NMN+ was 5-fold higher than that of NAD+ and has the greatest reported kcat of any substrate for CD38. 7-deaza-NHD+ was hydrolyzed at approximately one-third the rate of NHD+ but does not form a cyclic product. These results establish that a cyclic intermediate is not required for substrate hydrolysis. The ratio of methanolysis to hydrolysis for cADPR and NAD+ catalyzed by CD38 increases linearly with MeOH concentration. Both reactions produce predominantly the β-methoxy riboside compound, with a relative nucleophilicity of MeOH to H2O of 11. These results indicate the existence of a stabilized cationic intermediate for all observed chemistries in the active site of CD38. The partitioning of this intermediate between cyclization, hydrolysis, and nicotinamide-exchange unites the mechanisms of CD38 chemistries. Steady-state and pre-steady-state parameters for the partition and exchange mechanisms allowed full characterization of the reaction coordinate. Stopped-flow methods indicate a burst of cGDPR formation followed by the steady-state reaction rate. A lag phase, which was NGD+ concentration dependent, was also observed. The burst size indicates that the dimeric enzyme has a single catalytic site formed by two subunits. Pre- steady-state quench experiments did not detect covalent intermediates. Nicotinamide hydrolysis of NGD+ precedes cyclization and the chemical quench decomposes the enzyme-bound species to a mixture of cyclic and hydrolysis products. The time dependence of this ratio indicated that nicotinamide bond- breakage occurs 4 times faster than the conversion of the intermediate to products. Product release is the overall rate-limiting step for enzyme reaction with NGD+. | en_US |
dc.language | eng | en_US |
dc.publisher | American Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistry | en_US |
dc.relation.ispartof | Biochemistry | en_US |
dc.subject.mesh | Adp-Ribosyl Cyclase | en_US |
dc.subject.mesh | Adenosine Diphosphate Ribose - Analogs & Derivatives - Chemistry | en_US |
dc.subject.mesh | Antigens, Cd | en_US |
dc.subject.mesh | Antigens, Cd38 | en_US |
dc.subject.mesh | Antigens, Differentiation - Chemistry | en_US |
dc.subject.mesh | Binding, Competitive | en_US |
dc.subject.mesh | Catalysis | en_US |
dc.subject.mesh | Cyclic Adp-Ribose | en_US |
dc.subject.mesh | Fluorescence Polarization | en_US |
dc.subject.mesh | Guanosine Diphosphate Sugars - Chemistry | en_US |
dc.subject.mesh | Humans | en_US |
dc.subject.mesh | Hydrolysis | en_US |
dc.subject.mesh | Kinetics | en_US |
dc.subject.mesh | Membrane Glycoproteins | en_US |
dc.subject.mesh | Methanol | en_US |
dc.subject.mesh | Nad - Analogs & Derivatives - Chemistry | en_US |
dc.subject.mesh | Nad+ Nucleosidase - Chemistry | en_US |
dc.subject.mesh | Niacinamide - Pharmacology | en_US |
dc.subject.mesh | Nicotinamide Mononucleotide - Chemistry | en_US |
dc.subject.mesh | Spectrometry, Fluorescence | en_US |
dc.subject.mesh | Substrate Specificity | en_US |
dc.title | The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries | en_US |
dc.type | Article | en_US |
dc.identifier.email | Lee, HC:leehc@hku.hk | en_US |
dc.identifier.authority | Lee, HC=rp00545 | en_US |
dc.description.nature | link_to_subscribed_fulltext | en_US |
dc.identifier.doi | 10.1021/bi981248s | en_US |
dc.identifier.pmid | 9748331 | - |
dc.identifier.scopus | eid_2-s2.0-0032558401 | en_US |
dc.relation.references | http://www.scopus.com/mlt/select.url?eid=2-s2.0-0032558401&selection=ref&src=s&origin=recordpage | en_US |
dc.identifier.volume | 37 | en_US |
dc.identifier.issue | 38 | en_US |
dc.identifier.spage | 13239 | en_US |
dc.identifier.epage | 13249 | en_US |
dc.identifier.isi | WOS:000076088200023 | - |
dc.publisher.place | United States | en_US |
dc.identifier.scopusauthorid | Sauve, AA=6603026242 | en_US |
dc.identifier.scopusauthorid | Munshi, C=7003972383 | en_US |
dc.identifier.scopusauthorid | Lee, HC=26642959100 | en_US |
dc.identifier.scopusauthorid | Schramm, VL=35509217100 | en_US |
dc.identifier.issnl | 0006-2960 | - |