The Calcium Signaling Group, Department of Biochemistry and Molecular Cell Biology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany.
The Calcium Signaling Group, Department of Biochemistry and Molecular Cell Biology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany.
Biochim Biophys Acta Mol Cell Res. 2019 Jul;1866(7):1189-1196. doi: 10.1016/j.bbamcr.2018.11.011. Epub 2018 Nov 22.
The NAD-glycohydrolase/ADP-ribosyl cyclase CD38 catalyzes the metabolism of nicotinamide adenine dinucleotide (NAD) to the Ca mobilizing second messengers ADP-ribose (ADPR), 2'-deoxy-ADPR, and cyclic ADP-ribose (cADPR). In the present study, we investigated binding and metabolism of NAD by a soluble fragment of human CD38, sCD38, and its catalytically inactive mutant by monitoring changes in endogenous tryptophan (Trp) fluorescence. Addition of NAD resulted in a concentration-dependent decrease in sCD38 fluorescence that is mainly caused by the Trp residue W189. Amplitude of the fluorescence decrease was fitted as one-site binding curve revealing a dissociation constant for NAD of 29 μM. A comparable dissociation constant was found with the catalytically inactive sCD38 mutant (K 37 μM NAD) indicating that binding of NAD is not significantly affected by the mutation. The NAD-induced decrease in Trp fluorescence completely recovered in case of sCD38. Kinetics of recovery was slowed down with decreasing temperature and sCD38 concentration and increasing NAD concentration demonstrating that recovery in fluorescence is proportional to the enzymatic activity of sCD38. Accordingly, recovery in fluorescence was not observed with the catalytically inactive mutant. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
NAD-糖基水解酶/ADP-核糖基环化酶 CD38 催化烟酰胺腺嘌呤二核苷酸 (NAD) 的代谢为钙动员的第二信使 ADP-核糖 (ADPR)、2'-脱氧-ADPR 和环 ADP-核糖 (cADPR)。在本研究中，我们通过监测内源性色氨酸 (Trp) 荧光的变化，研究了可溶性人 CD38 片段 sCD38 及其催化失活突变体与 NAD 的结合和代谢。NAD 的加入导致 sCD38 荧光的浓度依赖性降低，这主要是由于色氨酸残基 W189 引起的。荧光降低的幅度拟合为单点结合曲线，揭示 NAD 的解离常数为 29μM。具有催化失活突变体的 sCD38 (K 37μM NAD) 也发现了类似的解离常数，表明 NAD 的结合不受突变的显著影响。在 sCD38 的情况下，NAD 诱导的 Trp 荧光降低完全恢复。随着温度、sCD38 浓度的降低和 NAD 浓度的增加，恢复荧光的动力学减慢，表明荧光的恢复与 sCD38 的酶活性成正比。因此，催化失活突变体没有观察到荧光恢复。本文是由 Claus Heizmann、Joachim Krebs 和 Jacques Haiech 编辑的特刊“ECS 会议”的一部分。
