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NAD binding by human CD38 analyzed by Trp189 fluorescence.

Valerie Wolters1, Anette Rosche1, Andreas Bauche1

  • 1The Calcium Signaling Group, Department of Biochemistry and Molecular Cell Biology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany.

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Summary

This study shows that nicotinamide adenine dinucleotide (NAD) binds to human CD38 with a dissociation constant of 29 μM. The enzyme

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • CD38 is an enzyme that metabolizes nicotinamide adenine dinucleotide (NAD).
  • NAD metabolism produces second messengers that mobilize calcium ions (Ca2+).
  • Key metabolites include ADP-ribose (ADPR) and cyclic ADP-ribose (cADPR).

Purpose of the Study:

  • To investigate the binding and metabolism of NAD by a soluble fragment of human CD38 (sCD38).
  • To characterize the interaction using a catalytically inactive sCD38 mutant.
  • To monitor these processes via changes in tryptophan fluorescence.

Main Methods:

  • Utilized intrinsic tryptophan fluorescence of sCD38 to monitor NAD binding.
  • Measured fluorescence changes upon NAD addition to wild-type and mutant sCD38.
  • Analyzed fluorescence decrease for binding kinetics and recovery for enzymatic activity.

Main Results:

  • NAD binding to sCD38 caused a concentration-dependent decrease in tryptophan fluorescence, primarily at W189.
  • Dissociation constant (KD) for NAD binding was determined to be 29 μM for sCD38.
  • Similar KD (37 μM) was observed for the catalytically inactive mutant, indicating mutation does not affect NAD binding.
  • Fluorescence decrease fully recovered with active sCD38, demonstrating enzymatic turnover; recovery was dependent on enzyme activity, temperature, and substrate concentration.
  • No fluorescence recovery was observed with the inactive mutant.

Conclusions:

  • NAD binds to sCD38 with high affinity, independent of catalytic activity.
  • The recovery of tryptophan fluorescence is a direct measure of CD38 enzymatic activity.
  • This study provides insights into the kinetics and mechanism of NAD metabolism by CD38.