title: Synthesis and properties of photoaffinity labels for the pyridine dinucleotide binding site of NAD glycohydrolase.
abstract: Two new photoactive analogues of oxidized nicotinamide adenine dinucleotide (NAD+) which are resistant to cleavage by NAD glycohydrolase were synthesized and characterized. The beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ was replaced with a 2,3-dihydroxycyclopentane ring forming a carbocyclic dinucleotide analogue. Photoreactivity was achieved by the incorporation of an azido group at the 8-position of the adenosyl ring. The previously published synthesis of carbocyclic pyridine dinucleotide analogues [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183] was modified by resolving the carbocyclic 1-aminoribose analogues and producing optically pure (+)-(1S)- or (-)-(1R)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. Each of these was converted to the corresponding carbocyclic nicotinamide 5'-nucleotide analogue and coupled with 8-azidoadenosine 5'-monophosphate. Two photoactive and isomeric NAD+ analogues were thus prepared. 8-Azidoadenosyl carba-NAD is the analogue in which D-dihydroxycyclopentane is substituted for the D-ribose of the nicotinamide nucleoside moiety. 8-Azido-adenosyl pseudocarba-NAD contains the L-carbocycle in place of the D-ribotide ring. 8-Azidoadenosyl carba-NAD was shown to inhibit the NAD glycohydrolase from Bungarus fasciatus venom competitively with an inhibitor dissociation constant of 187 microM. 8-Azidoadenosyl pseudocarba-NAD was shown to inhibit the same enzyme competitively with a Ki of 73 microM. The superior NADase inhibitor, 8-azidoadenosyl pseudocarba-NAD, was characterized kinetically and shown to fulfill the criteria required of a specific active site directed photoaffinity probe. Irradiation of mixtures of the photoprobe and NAD glycohydrolase with short-wave ultraviolet light resulted in the rapid and irreversible loss of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
authors.forename: J T
authors.initials: JT
authors.lastname: Slama
authors.forename: A M
authors.initials: AM
authors.lastname: Simmons
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D004546:Elapid Venoms; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009904:Optical Rotation; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000345:Affinity Labels; D004546:Elapid Venoms; D007202:Indicators and Reagents; D009243:NAD; D009244:NAD+ Nucleosidase