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Article: The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries

TitleThe reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries
Authors
Issue Date1998
PublisherAmerican 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?
AbstractHuman 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 Identifierhttp://hdl.handle.net/10722/171658
ISSN
2015 Impact Factor: 2.876
2015 SCImago Journal Rankings: 1.769
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorSauve, AAen_US
dc.contributor.authorMunshi, Cen_US
dc.contributor.authorLee, HCen_US
dc.contributor.authorSchramm, VLen_US
dc.date.accessioned2012-10-30T06:16:12Z-
dc.date.available2012-10-30T06:16:12Z-
dc.date.issued1998en_US
dc.identifier.citationBiochemistry, 1998, v. 37 n. 38, p. 13239-13249en_US
dc.identifier.issn0006-2960en_US
dc.identifier.urihttp://hdl.handle.net/10722/171658-
dc.description.abstractHuman 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.languageengen_US
dc.publisherAmerican Chemical Society. The Journal's web site is located at http://pubs.acs.org/biochemistryen_US
dc.relation.ispartofBiochemistryen_US
dc.subject.meshAdp-Ribosyl Cyclaseen_US
dc.subject.meshAdenosine Diphosphate Ribose - Analogs & Derivatives - Chemistryen_US
dc.subject.meshAntigens, Cden_US
dc.subject.meshAntigens, Cd38en_US
dc.subject.meshAntigens, Differentiation - Chemistryen_US
dc.subject.meshBinding, Competitiveen_US
dc.subject.meshCatalysisen_US
dc.subject.meshCyclic Adp-Riboseen_US
dc.subject.meshFluorescence Polarizationen_US
dc.subject.meshGuanosine Diphosphate Sugars - Chemistryen_US
dc.subject.meshHumansen_US
dc.subject.meshHydrolysisen_US
dc.subject.meshKineticsen_US
dc.subject.meshMembrane Glycoproteinsen_US
dc.subject.meshMethanolen_US
dc.subject.meshNad - Analogs & Derivatives - Chemistryen_US
dc.subject.meshNad+ Nucleosidase - Chemistryen_US
dc.subject.meshNiacinamide - Pharmacologyen_US
dc.subject.meshNicotinamide Mononucleotide - Chemistryen_US
dc.subject.meshSpectrometry, Fluorescenceen_US
dc.subject.meshSubstrate Specificityen_US
dc.titleThe reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistriesen_US
dc.typeArticleen_US
dc.identifier.emailLee, HC:leehc@hku.hken_US
dc.identifier.authorityLee, HC=rp00545en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1021/bi981248sen_US
dc.identifier.pmid9748331-
dc.identifier.scopuseid_2-s2.0-0032558401en_US
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0032558401&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume37en_US
dc.identifier.issue38en_US
dc.identifier.spage13239en_US
dc.identifier.epage13249en_US
dc.identifier.isiWOS:000076088200023-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridSauve, AA=6603026242en_US
dc.identifier.scopusauthoridMunshi, C=7003972383en_US
dc.identifier.scopusauthoridLee, HC=26642959100en_US
dc.identifier.scopusauthoridSchramm, VL=35509217100en_US

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