The Double pH Optimum of 5'-Nucleotidase of Bull Seminal Plasma

The action of bull seminal plasma 5'-nucleotidase on adenosine 5'-phosphate in the presence of magnesium has one optimum at pH 7.5 to 8.0 and a second at pH 9.1 to 9.3. Only one optimum, at pH 7.5 to 8.0, occurs in the absence of magnesium. The phenomenon of a double pH optimum was independent of the nature of the buffer that was employed. A magnesium concentration of 0.005 to 0.01 m and a temperature of at least 25° were necessary for the appearance of the second pH optimum. The apparent energy of activation in the absence or presence of magnesium was dependent on pH. Maximal values of 10,300 cal per mole at pH 7.5 to 8.0 and 15,400 cal per mole at pH 9.2 were obtained in the absence and presence of magnesium, respectively. All nine nucleotides that were investigated in the absence of magnesium showed only one pH optimum at approximately 7.5 to 8.0. In the presence of magnesium, all nucleotides manifested an optimum at pH 9.0 to 9.3; of these the 5'-phosphates of adenosine, guanosine, and inosine also showed a pH optimum at approximately 7.5. The apparent K m values for deoxyribonucleotides were in general much larger than those for the corresponding ribonucleotides. In the absence of Mg ++ , the apparent pK m values for the action of 5'-nucleotidase on adenosine 5'-phosphate were constant, within experimental variation, from pH 7.0 to pH 8.7, and thereafter decreased with a slope of -1. In the presence of Mg ++ the apparent pK m values were constant until a pH of approximately 9.5 and thereafter decreased with the same slope. A formulation is proposed for the phenomenon of the double pH optimum in the action of 5'-nucleotidase, and for the influence of Mg ++ on this effect. Four sites are postulated to be involved in the interaction of the enzyme and the substrate; the role of each of these sites is discussed.