Hecht S M, Hawrelak S D
Biochemistry. 1975 Mar 11;14(5):974-81. doi: 10.1021/bi00676a016.
Rye grass 3'-nucleotidase has been purified to apparent homogeneity on Sephadex A-25 and CM-cellulose columns and shown to hydrolyze 2'-O-methyladenosine 3'-monophohate and 2'-deoxyadenosine 3'-monophosphate 35.8 and 542 times more slowly than the normal substrate (3'-AMP), verifying the importance of the 2'-beta-OH group of the substrate in the overall hydrolysis process. Although neither was hydrolyzed as rapidly as 3'-AMP, both the 2'-O-methyl and 2'-deoxy analogs acted as competitive inhibitors of the hydrolysis of 3'-AMP (K-m equals 0.12 mM), with apparent K-i's of 0.39 and 0.51 nM, respectively. In order to determine the possible susceptibility of naturally occurring ribonucleoside 3'-diphosphates, such as guanosine tetraphosphate (ppGpp), to 3'-phosphohydrolase activities, the 3'-nucleotidase was also employed in the attempted pyrophosphorolysis of adenosine 3'-diphosphate and guanosine tetraphosphate. Neither adenosine 3'-diphosphate nor guanosine tetraphosphate was degraded at a significant rate by the nucleotidase, relative to the normal substrate.
黑麦草3'-核苷酸酶已通过葡聚糖凝胶A - 25和CM - 纤维素柱纯化至表观均一，并显示其水解2'-O - 甲基腺苷3'-单磷酸和2'-脱氧腺苷3'-单磷酸的速度分别比正常底物（3'-AMP）慢35.8倍和542倍，这证实了底物的2'-β-OH基团在整个水解过程中的重要性。尽管两者的水解速度都不如3'-AMP快，但2'-O - 甲基和2'-脱氧类似物均作为3'-AMP水解的竞争性抑制剂（K - m等于0.12 mM），表观K - i分别为0.39和0.51 nM。为了确定天然存在的核糖核苷3'-二磷酸，如鸟苷四磷酸（ppGpp）对3'-磷酸水解酶活性的可能敏感性，3'-核苷酸酶还用于尝试对腺苷3'-二磷酸和鸟苷四磷酸进行焦磷酸解。相对于正常底物，腺苷3'-二磷酸和鸟苷四磷酸均未被核苷酸酶以显著速率降解。
