Activity of IMP- and AMP-preferring isoforms of 5'-nucleotidase from human seminal plasma with AMP analogues.

AMP analogues modified at various positions of the molecule were checked as substrates for the two soluble isoforms of 5'-nucleotidase from human seminal plasma. These isoforms were isolated to near homogeneity by affinity chromatographies. AMP derivatives were differently dephosphorylated by both the isoforms depending on the site of modification in the natural compound. Changes in the phosphate moiety reduced significantly hydrolysis by the IMP-preferring form, whereas the AMP-preferring form was less affected. The AMP-preferring form was characterized by a relatively broad specificity toward substrate analogues indicating that the binding domains for the phosphate moiety of these isoforms are not identical. Substitutions at the C-8 adenine base reduced the hydrolysis rate of both the enzymes and variations of the syn-anti conformational equilibrium resulted in different effects on catalysis by both forms. Therefore, the orientation of the heterocyclic base around the glycosidic bond may not be the crucial factor affecting binding and catalytic activity. Hydrogen bonding potential of base N-7 was essential for the binding and catalysis of the IMP- but not of the AMP-preferring form. This was the most striking difference between the studied isoforms. Modifications and substitutions of 6-amino function, better accepted by the IMP-preferring form than by the AMP-preferring form, indicated that no essential hydrogen bonding is required for catalytic activity. The binding was however significantly slowed in 6-SH-PuMP. Hydrogen bonding potential of N-1 was significant for the hydrolysis rate of the IMP- but not of the AMP-preferring form. We suggest that these human seminal plasma isoforms of soluble 5'-nucleotidase, characterized by unique features, may represent the tissue-specific expression of the polymorphic gene.