Purine nucleoside phosphorylase: Inhibition by purine N(7)- and N(9)-acyclonucleosides; and substrate properties of 7-β- D-ribofuranosylguanine and 7-β- D-ribofuranosylhypoxanthine

Abstract

A series of 10 N(9)- and N(9)-acyclonucleosides of guanine and 8-substituted guanines (8-Br, 8 8-SH, and 8-NH 2), 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 K i = 5 vs 14 μM for the human enzyme and 0.7 vs 2.3 μM 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-β- D-ribosides of guanine (N 7Guo) and hypoxanthine (N 7Ino) were weak substrates of PNP from human erythrocytes, calf spleen and E. coli. With the human enzyme the pseudo-first-order rate constants ( V max/ K m ) for phosphorolysis of N 7Guo and N 7Ino were 0.08 and 0.02% that for Ino. The Michaelis constants ( K m for N 7Guo were 27 (calf PNP), 108 (human PNP) and 450 -gmM ( E. coli PNP). For N 7Ino the corresponding K m 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 phosphoolysis of N 7Ino 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, 9212-9-9217, 1988), were examined in relation to present concepts regarding binding of substrates and inhibitors at the active site(s) of these enzymes.