Structure-activity relationship of cytoplasmic 5′-nucleotidase substrate sites

Abstract

Various 5'-nucleotidases (EC 3.1.3.5) exist in vertebrate tissues. The sequence and cDNA cloning of the membrane-bound ecto-5'-nucleotidase (e-N) and one of the cytosolic isoenzymes, IMP-preferring (c-N-II), but not the cytosolic AMP-preferring form (c-N-I), have been reported. While c-N-II has a broad tissue distribution, c-N-I is found only in vertebrate heart. The published data on substrate specificity involve mainly the naturally occurring nucleoside monophosphates, without a systematic structure-activity relationship study. In the present study we have used a series of AMP and IMP analogues to examine the structure-activity relationship for c-N-I and c-N-II in detail. The rank order of activity of the test compounds differed substantially between c-N-I and c-N-II. c-N-I and c-N-II varied with respect to the following interactions with substrate: (1) hydrogen-bond formation with the substituent in the 6-position of the purine ring (a donor-type with c-N-I and an acceptor-type with c-N-II); and (2) hydrophobic attraction of the 6-position unsubstituted purine ring (more pronounced with c-N-I than with c-N-II). No better substrate than 5'-AMP was found for c-N-I. We propose that c-N-I functions as an AMP-binding protein in the myocardial cell with an important role during ischaemic ATP breakdown when AMP accumulates rapidly.

Thiopurine Metabolism and Identification of the Thiopurine Metabolites Transported by MRP4 and MRP5 Overexpressed in Human Embryonic Kidney Cells

“…5A), followed by a rise in MPrib. This suggests that the MPrib is made by dephosphorylation of tIMP by a phosphatase or nucleotidase (Skladanowski et al, 1996;Hunsucker et al, 2001), followed by rapid excretion of the nucleoside via a nucleoside transporter (Baldwin et al, 1999). As expected, intracellular tXMP and TXrib concentrations follow the rise in tIMP in the cells with some delay (Fig.…”

“…Any mechanism that reduces the intracellular concentration of tGMP may act as a potential resistance mechanism. As mentioned above, 5Ј-nucleotidase reduces the tNMP pool and has been linked to thiopurine resistance (Rosman et al, 1974;Skladanowski et al, 1996). Furthermore, TPMT methylates 6MP, forming MeMP, which reduces the 6MP concentration and toxicity (Lennard et al, 1987).…”

5'-Nucleotidases, Nucleosides and their Distribution in the Brain: Pathological and Therapeutic Implications

“…The substrate specificity of 5´NTs is based on its recognition of the nucleobase. Not surprisingly, hydrogen-bond formation with the substituent in the 6-position of the purine ring and the hydrophobic attractions play major roles in the substrate specificity of 5´NTs [164]. The electron pair on N-1 is important in substrate binding of cN-II but is not a prerequisite for cN-I.…”

Partial characterization of 5′-nucleotidase from giant squid (Dosidicus gigas) mantle Caracterización parcial de la enzima 5′-nucleotidasa del manto de calamar gigante (Dosidicus gigas)

“…Likewise, human 5 0 -nucleotidase activity was affected by ADP in range concentrations from 0 to 2 mM, increasing its specific activity from 1 to 38 mM Pi/min/mg protein (Hunsucker et al, 2001). On the other hand, when Skladanowski et al (1996) were working with 5 0 -nucleotidase from rat they observed an ADP activator effect in range from 0 to 1 mM, in which its specific activity increased from 0.1 to 0.3 U/ mg. Nevertheless, both ATP and ADP may also act as inhibitors depending on the enzyme source (Naito & Lowenstein, 1981).…”

“…Skladanowski, Hoffmann, Krass, Jastorff, and Makarewicz (1996) detected a biphasic behavior in purified enzyme of pigeon heart, 5 0 -nucleotidase was activated in the range from 0 to 500 mM with a max peak of 50 mM. Willadsen, Nielsen, and Riding (1989) reported 70% Figure 3.…”