Enzymatic assays for NAD-dependent deacetylase activities

Area of Science:

• Biochemistry
• Enzymology

Background:

• NAD-dependent deacetylases are a novel enzyme class catalyzing protein deacetylation.
• These enzymes utilize a reactive ADP-ribose intermediate, not water, for acetyl group transfer.
• Key reaction products include nicotinamide, acetyl-ADP-ribose, and deacetylated substrates.

Purpose of the Study:

• To review and present various assays for measuring NAD-dependent deacetylase activity.
• To cover methods for both deacetylation and NAD hydrolysis reactions catalyzed by these enzymes.
• To guide researchers in selecting appropriate assays for studying these enzymes.

Main Methods:

• Preparation of acetylated protein and peptide substrates for deacetylation assays.
• Description of four deacetylation assays, including direct measurement of acetyl group loss and acetate production.
• Presentation of two indirect assays: NAD hydrolysis and a novel NAD-nicotinamide exchange reaction.
• Detailed explanation of a quantitative method using HPLC for product measurement with a monoacetylated peptide substrate.

Main Results:

• Established methods for substrate preparation are presented.
• Multiple direct and indirect assay strategies for NAD-dependent deacetylases are detailed.
• A quantitative HPLC-based method offers precise product measurement.

Conclusions:

• A comprehensive overview of available assays for NAD-dependent deacetylases is provided.
• The review facilitates the study and characterization of this important enzyme class.
• Researchers can utilize these diverse assay methods to investigate enzyme kinetics and mechanisms.