Kinetic isotope effect studies on the formation and hydrolysis of cADPR by CD38A

A. Sauve, C. H. Lee, Vern L. Schramm

Research output: Contribution to journalArticle

Abstract

The lymphocyte protein ('I)38 has been shown lo Gill alvze tile trormation of cyclic ADP ribose (cADPR) from NAD+ and lo hydrolvze cADPtt and NAD+ to ADP-ribose (.DPR). The goals of this projecl are to characterize the three transition state structures for the three chemically distincl reactions catalyzed by CD38. Kinetic isotope el[acts tRIEs tirh, drolysis of radiolabeh'd sub strates cADPR and NAD+ were determined at 37, of and pll 7.5 in 50 mM phosphate buffer for soluble recombinant (7I)38. Kldestaer hevdrolysis of NAD+ labeled with l'-:ttl and l' HC were 1.010 and too respectively, demonstrating that chemistry is not rate limiting for the reaction, l,abeled cADPR was synthesized with ADP-ribosylcyclase and isotopically labeled NAD+. Kinetic isotope effects obtained for cADPR hydrolysis indicated rate-limiting chemistry by the relalively large KIEs of tilt (1.26). 1-24 (1.050). and 5'-61 (l.051). The modest iLI4 KIE arid the large are consistent with an oxocarbenium-ion transition state. Studies el methanolysis of NAD+ and cADIR suggest that a stabilized cation at the active, lite precedes ADPI/for marion. A relative nucleophilicily of 10/l: MeOII/H20 in the product ratio for methoxy-ADPR/A1)PR for both reactions suggests an intermediate lifetime which dermits solvent equilibration. Only' .#methoxy ADPII. was detected as a product of methanolysis indicating that hydrolysis likely proceeds with retention of configuration at (21'. The methanolysis results implicate a common intermediate toward ADPIC formation starting from till her.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1997

Fingerprint

Cyclic ADP-Ribose
ribose
Isotopes
NAD
Hydrolysis
isotopes
methanolysis
hydrolysis
kinetics
Kinetics
chemistry
Adenosine Diphosphate Ribose
tiles
Lymphocytes
Tile
cations
gills
lymphocytes
buffers
Adenosine Diphosphate

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Kinetic isotope effect studies on the formation and hydrolysis of cADPR by CD38A. / Sauve, A.; Lee, C. H.; Schramm, Vern L.

In: FASEB Journal, Vol. 11, No. 9, 1997.

Research output: Contribution to journalArticle

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