Purine catabolism in man: inhibition of 5′-phosphomonoesterase activities from placental microsomes

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Abstract
The 5''-phosphomonoesterase activity of 5''-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.1) participates in the catabolism of purine ribonucleotides to uric acid in humans. Initial velocity studies of 5''-nucleotidase suggest a sequential mechanism of interaction between AMP and MgCl2, with a Km of 14 and 3 .mu.M, respectively. With product inhibition studies the apparent Ki for adenosine, inosine, cytidine and inorganic phosphate were 0.4, 3.0, 5.0 and 42 mM, respectively. A large number of nucleoside mono, di and tri-phosphate compounds were inhibitors of the enzyme. Allopurinol ribonucleotide, ADP or ATP were competitive inhibitors when AMP was the substrate, with a Ki slope of 10, 20 or 54 .mu.M, respectively. GTP was a noncompetitive inhibitor, with a Ki slope of 120 .mu.M. The phosphomonoesterase activity of human placental microsomal alkaline phosphatase had a pH optimum of 10.0 and had only 18% of maximum activity at pH 7.4. Substrates and inhibitors included almost any phosphorylated compound. The Km for AMP was 0.4 mM and the apparent Ki for Pi was 0.6 mM. Activity was increased only 19% by 5 mM MgCl2. 5''-Nucleotidase and alkaline phosphatase may be inhibited by ATP and Pi respectively, under normal intracellular conditions, and AMP may be preferentially hydrolyzed by 5''-nucleotidase.