ATP and phosphate reciprocally affect subunit association of human recombinant High Km 5 '-nucleotidase - Role for the C-terminal polyglutamic acid tract in subunit association and catalytic activity

IMP-specific, High Km 5'-nucleotidase (EC 3.1.3.5) is an ubiquitous enzyme, the activity of which is highly regulated by substrate, ATP, and inorganic phosphate. The cDNA encoding this enzyme has recently been cloned and foun d to contain a unique stretch of nine glutamic and four aspartic acid resid ues at the C-terminus. To study the effects of this acidic tail, and of ATP and inorganic phosphate on enzyme function, we generated several structura l modifications of the 5'-nucleotidase cDNA, expressed the corresponding pr oteins in Escherichia coli and compared their molecular and kinetic propert ies. As with the enzyme purified from human placenta, all recombinant prote ins were activated by ATP and inhibited by inorganic phosphate. Although th e S-0.5-values were higher, the specific activities of the purified protein variants (except that truncated at the C-terminus) were similar. The molec ular mass of the full-length enzyme subunit has been estimated at 57.3 kDa and the molecular mass of the native protein, as determined by gel-filtrati on chromatography, was estimated to be 195 kDa. Increasing the concentratio n of NaCl to 0.3 M promoted oligomerization of the protein and the formatio n of aggregates of 332 kDa. ATP induced further oligomerization to 715 kDa, while inorganic phosphate reduced the estimated molecular mass to 226 kDa. In contrast to the truncation of 30 amino acids at the N-terminus, which d id not alter enzyme properties, the removal of the polyglutamic/aspartic ac id tail of 13 residues at the C-terminus caused profound kinetic and struct ural changes, including a 29-fold decrease in specific activity and a signi ficant increase in the sensitivity to inhibition by inorganic phosphate in the presence of AMP. Structurally, there was a dramatic loss of the ability to form oligomers at physiological salt concentration which was only parti ally restored by the addition of NaCl or ATP. These data suggest an importa nt function of the polyglutamic acid tract in the process of association an d dissociation of 5'-nucleotidase subunits.