Amino acid-specific ADP-ribosylation.

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• ADP-ribosylation is a post-translational modification involving the transfer of ADP-ribose from NAD+ to target proteins.
• The chemical nature of ADP-ribosyl linkages can vary, influencing their stability and biological function.
• Previous studies have described "hydroxylamine-stable" and "hydroxylamine-labile" bonds in ADP-ribosylation, but their chemical basis was unclear.

Purpose of the Study:

• To synthesize and characterize ADP-ribose conjugates formed by an NAD:arginine ADP-ribosyltransferase.
• To investigate the stability of ADP-ribosyl-agmatine and ADP-ribosyl-histone linkages under different chemical conditions.
• To elucidate the chemical basis for the differential stability of ADP-ribosyl bonds to hydroxylamine.

Main Methods:

• Synthesis of radiolabeled ADP-ribose-agmatine and ADP-ribose-histone using [14C]NAD+.
• Chemical treatment of synthesized conjugates with sodium hydroxide and neutral hydroxylamine.
• Analysis of reaction products using high-pressure liquid chromatography (HPLC) and complexation with ferric chloride.
• Enzymatic hydrolysis of ADP-ribose-hydroxamate with snake venom phosphodiesterase.

Main Results:

• Identical pseudo-first order rate constants for the breakdown of ADP-ribose-agmatine and ADP-ribose-histone in NaOH and hydroxylamine.
• Hydroxylamine treatment yielded ADP-ribose-hydroxamate, confirmed by HPLC and ferric chloride complex formation.
• Snake venom phosphodiesterase hydrolysis of ADP-ribose-hydroxamate produced 5 '-AMP, indicating a pyrophosphate bond.
• ADP-ribosyl-protein linkage formed by the NAD:arginine ADP-ribosyltransferase is more stable to hydroxylamine than the ADP-ribose-glutamate bond.

Conclusions:

• The ADP-ribosyl-arginine linkage is significantly more stable to hydroxylamine than the ADP-ribose-glutamate linkage.
• These findings provide a chemical explanation for the "hydroxylamine-stable" and "hydroxylamine-labile" bonds observed in ADP-ribosylation.
• The differential stability is attributed to the specific amino acid residue involved in the ADP-ribosyl linkage.