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

Journal Article · · Journal of Biological Chemistry; (USA)
OSTI ID:7191918
;  [1]
  1. Univ. of Kentucky, Lexington (USA)
+ Show Author Affiliations
A photoactive coenzyme analog of NAD+ has been synthesized by chemically coupling ({sup 32}P)2-azido-AMP and NMN to produce ({sup 32}P)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.
OSTI ID:
7191918
Journal Information:
Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:7; ISSN JBCHA; ISSN 0021-9258
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
AMINO ACIDS
AMP
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARBOXYLIC ACIDS
CATTLE
CHEMICAL PREPARATION
CHEMICAL PROPERTIES
COENZYMES
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ELECTROMAGNETIC RADIATION
ENZYME ACTIVITY
ENZYMES
GLANDS
GLUTAMIC ACID
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIVER
MAMMALS
NAD
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
OXIDOREDUCTASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIATIONS
RADIOISOTOPES
REAGENTS
RUMINANTS
SPECTROPHOTOMETRY
SYNTHESIS
TRACER TECHNIQUES
ULTRAVIOLET RADIATION
VERTEBRATES