Bzowska A, Ananiev A V, Ramzaeva N, Alksins E, Maurins J A, Kulikowska E, Shugar D
Department of Biophysics, University of Warsaw, Poland.
Biochem Pharmacol. 1994 Aug 30;48(5):937-47. doi: 10.1016/0006-2952(94)90364-6.
A series of 10 N(7)- and N(9)-acyclonucleosides of guanine and 8-substituted guanines (8-Br, 8-SH and 8-NH2), and two N(7)-acyclonucleosides of hypoxanthine, were tested for their ability to inhibit purine nucleoside phosphorylase (PNP) (E.C. 2.4.2.1) from human erythrocytes and rabbit kidney. The acyclic chains contained a nitrogen in place of a carbon at the 3', 4' or 5' position and, in one case, an ether oxygen at the 2' position. Most striking was the finding that one of the N(7)-acyclonucleoside analogues, 7-[(1,3-dihydroxypropyl-2)amino]ethylguanine, proved to be a 3-fold more effective inhibitor than its corresponding N(9) counterpart, with Ki = 5 vs 14 microM for the human enzyme and 0.7 vs 2.3 microM for the rabbit enzyme. Both analogues, as well as the others examined, inhibited phosphorolysis competitively with respect to nucleoside substrates (inosine with the human enzyme and guanosine with the rabbit enzyme). The foregoing logically led to the finding that the 7-beta-D-ribosides of guanine (N7Guo) and hypoxanthine (N7Ino) were weak substrates of PNP from human erythrocytes, calf spleen and E. coli. With the human enzyme the pseudo-first-order rate constants (Vmax/Km) for phosphorolysis of N7Guo and N7Ino were 0.08 and 0.02% that for Ino. The Michaelis constants (Km) for N7Guo were 27 (calf PNP), 108 (human PNP) and 450 microM (E. coli PNP). For N7Ino the corresponding Km values were 1.52, 1.26 and 0.64 mM. Four previously well-characterized N(9)-acyclonucleoside inhibitors of calf spleen PNP were found to inhibit phosphorolysis of N7Ino by the same enzyme 2-10-fold more effectively than the parent Ino. The overall results, along with the known excellent substrate properties of N(7)-alkyl- Guo and Ino (Bzowska et al. J Biol Chem 263, 9212-9217, 1988), were examined in relation to present concepts regarding binding of substrates and inhibitors at the active site(s) of these enzymes.
对一系列10种鸟嘌呤和8 - 取代鸟嘌呤（8 - Br、8 - SH和8 - NH₂）的N(7)-和N(9)-无环核苷，以及2种次黄嘌呤的N(7)-无环核苷，测试了它们抑制人红细胞和兔肾中嘌呤核苷磷酸化酶（PNP，E.C. 2.4.2.1）的能力。无环链在3'、4'或5'位含有一个氮原子取代碳原子，在一种情况下，在2'位含有一个醚氧原子。最引人注目的发现是，一种N(7)-无环核苷类似物，7 - [(1,3 - 二羟基丙基 - 2)氨基]乙基鸟嘌呤，被证明是其相应N(9)类似物的3倍有效抑制剂，对人酶的Ki值分别为5 μM和14 μM，对兔酶的Ki值分别为0.7 μM和2.3 μM。这两种类似物以及其他所检测的类似物，相对于核苷底物（人酶作用下的肌苷和兔酶作用下的鸟苷）竞争性抑制磷酸解作用。上述情况合乎逻辑地导致发现鸟嘌呤（N7Guo）和次黄嘌呤（N7Ino）的7 - β - D - 核糖苷是人红细胞、小牛脾脏和大肠杆菌中PNP的弱底物。对于人酶，N7Guo和N7Ino磷酸解的伪一级速率常数（Vmax/Km）分别是肌苷的0.08%和0.02%。N7Guo的米氏常数（Km）分别为27（小牛PNP）、108（人PNP）和450 μM（大肠杆菌PNP）。对于N7Ino，相应的Km值分别为1.52、1.26和0.64 mM。发现4种先前已充分表征的小牛脾脏PNP的N(9)-无环核苷抑制剂抑制该酶对N7Ino的磷酸解作用比母体肌苷有效2 - 10倍。结合关于这些酶活性位点上底物和抑制剂结合的现有概念对总体结果以及已知的N(7)-烷基 - Guo和Ino的优异底物特性（Bzowska等人，《生物化学杂志》263, 9212 - 9217, 1988）进行了研究。
