Synthesis and properties of 2-azido-NAD+. A study of interaction with glutamate dehydrogenase

• Published in: The Journal of biological chemistry Vol. 265; no. 7; pp. 3636 - 3641
• Main Authors: Kim, H, Haley, B E
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.03.1990
• Subjects: 550201 - Biochemistry- Tracer Techniques; AFFINITY; Affinity Labels - chemical synthesis; AMINO ACIDS; AMP; Analytical, structural and metabolic biochemistry; ANIMALS; Azides - chemical synthesis; Azides - metabolism; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; Binding Sites; Biological and medical sciences; BODY; CARBOXYLIC ACIDS; CATTLE; CHEMICAL PREPARATION; CHEMICAL PROPERTIES; COENZYMES; Coenzymes, vitamins; DAYS LIVING RADIOISOTOPES; DIGESTIVE SYSTEM; DOMESTIC ANIMALS; ELECTROMAGNETIC RADIATION; ENZYME ACTIVITY; ENZYMES; Fundamental and applied biological sciences. Psychology; GLANDS; Glutamate Dehydrogenase - metabolism; GLUTAMIC ACID; Indicators and Reagents; Isomerism; ISOTOPE APPLICATIONS; ISOTOPES; LIGHT NUCLEI; LIVER; Liver - enzymology; MAMMALS; NAD; NAD - chemical synthesis; NAD - metabolism; NUCLEI; NUCLEOTIDES; ODD-ODD NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANS; Other biological molecules; OXIDOREDUCTASES; PHOSPHORUS 32; PHOSPHORUS ISOTOPES; RADIATIONS; RADIOISOTOPES; REAGENTS; RUMINANTS; SPECTROPHOTOMETRY; SYNTHESIS; TRACER TECHNIQUES; ULTRAVIOLET RADIATION; Ultraviolet Rays; VERTEBRATES; Glutamate dehydrogenase; Bovine; Photoactivation; Liver; Active site; Dissociation constant; Molecular interaction; Coenzyme; Azido complex; NAD; Binding protein; Vertebrata; Mammalia; Enzymatic activity; Analog; Molecular probe; Artiodactyla; Inhibition; Chemical synthesis; Ungulata
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)39640-1

A photoactive coenzyme analog of NAD+ has been synthesized by chemically coupling [32P]2-azido-AMP and NMN to produce [32P]nicotinamide 2-azidoadenosine dinucleotide (2-azido-NAD+). The utility of 2-azido-NAD+ as an effective active-site-directed photoprobe was demonstrated using bovine liver glutamate dehydrogenase as a model enzyme. In the absence of ultraviolet light, 2-azido-NAD+ is a substrate for this enzyme. Photoincorporation of probe was saturable with two different apparent dissociation constants of 10 microM and 40 microM. Protection of photoinsertion was seen with the natural substrate NAD+ with apparent dissociation constants of less than 5 microM and 25 microM. This observation may be explained on the basis of negative cooperative interaction between the subunits. The photoinsertion of 2-azido-NAD+ was increased by GTP and decreased by ADP in accordance with their known effects on NAD+ binding. When the enzyme was covalently modified by photolysis in the presence of saturating amounts of photoprobe, an approximately 40% inhibition of the enzyme activity was observed. These results demonstrate that the photoaffinity coenzyme analog has potential application as a probe to characterize NAD(+)-binding proteins and to identify the active sites of these proteins.