NAD Synthetase is responsible for the final step in the synthesis of Nicotinamide Adenine Dinucleotide (NAD). The enzyme sourced from Escherichia coli exhibits Km values of 200 μM for NAD, 11 μM for ATP, and 0.65 μM for ammonia. Conversely, NAD Synthetase derived from yeast demonstrates Km values of 170 μM for NAD, 190 μM for ATP, and 64 μM for ammonia. This enzyme is applicable in enzymatic assays for ATP, ammonia, urea, or creatinine and can also be used in enzymatic recycling methods. Additionally, NAD Synthetase holds potential for research into metabolic diseases, cancer, aging, and neurodegenerative disorders.