Synthesis and properties of photoaffinity labels for the pyridine dinucleotide binding site of NAD glycohydrolase.
Synthesis and properties of photoaffinity labels for the pyridine dinucleotide binding site of NAD glycohydrolase.
复制标题
NAD 糖水解酶吡啶二核苷酸结合位点的光亲和标记的合成和特性。
DOI：
10.1021/bi00223a033
复制
发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Simmons,AM
中科院分区：
文献类型：
--
作者：
Slama,JT;Simmons,AM
摘要：合成了两种新的抗NAD糖水解酶裂解的氧化烟酰胺腺嘌呤二核苷酸（NAD+）光活性类似物并对其进行了表征。/ ?NAD+的烟酰胺核苷部分的-d核糖核苷酸环被2,3 -二羟基环戊烷环取代，形成碳环二核苷酸类似物。通过在腺苷环的8位加入一个叠氮基团实现光反应性。先前发表的碳环吡啶二核苷酸类似物的合成[Slama， J. T., & Simmons， A. M.(1988) Biochemistry 27,183]通过分解碳环1-氨基类似物并产生光学纯的(+)-(lS)-或(-)-（l/?）4/3-amino-2a 3 a-dihydroxy-l / 3-cyclopentanemethanol。每一种都转化为相应的碳环烟酰胺S'-核苷酸类似物，并与8-叠氮腺苷S'-单磷酸偶联。因此制备了两种具有光活性和异构体的NAD+类似物。8-叠氮腺苷碳- nad是用d -二羟基环戊烷取代烟酰胺核苷部分的d -核糖的类似物。8-叠氮腺苷伪碳- nad含有l -碳环代替d -核糖体环。8-Azidoadenosyl carba-NAD可竞争性地抑制Bungarus fasciatus venom的NAD糖水解酶，解离常数为187µ。8-Azidoadenosyl pseudocarba-NAD对该酶具有竞争性抑制作用，Kx为73µ。该NADase抑制剂8-叠氮腺苷基伪碳水化合物- nad经过动力学表征，符合特定活性位点定向光亲和探针的要求。光探针与NAD糖水解酶的混合物在短波紫外光照射下，酶活性迅速且不可逆地丧失。无论是光探针在黑暗中还是在缺乏叠氮基团的类似物的酶溶液的照射下，都没有观察到时间依赖性的酶抑制作用。在底物NAD+和非光活性竞争抑制剂的存在下，光失活速率减慢，并在低浓度的光探针下饱和。
Revised Manuscript Received November 30, 1990 abstract: Two new photoactive analogues of oxidized nicotinamide adenine dinucleotide (NAD+) which are resistant to cleavage by NAD glycohydrolase were synthesized and characterized. The/?-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-aminoriboseanalogues and producingoptically pure (+)-(lS)-or (-)-(l/?)-4/3-amino-2a, 3a-dihydroxy-l/3-cyclopentanemethanol. Each of these was converted to the corresponding carbocyclic nicotinamide S'-nucleotide analogue and coupled with 8-azidoadenosine S'-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 µ. 8-Azidoadenosyl pseudocarba-NAD was shown to inhibit the same enzyme competitively with a Kx of 73 µ. 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 ultravioletlight resulted in the rapid and irreversible loss of enzyme activity. No time-dependent enzyme inhibition was observed either with the photoprobe in the dark or on irradiation of solutions of the enzyme with analogues lacking the azido group. Rates of photoinactivation were slowed in the presence of the substrate NAD+ and in the presence of nonphotoactive competitive inhibitors and were shown to saturate at low concentrations of photoprobe.