Abstract
1. Inorganic phosphate was liberated from bis-p-nitrophenyl phosphate by Fusarium acid phosphatase II at half the rate observed for the liberation of p-nitrophenol. No time lag was observed for the liberation of inorganic phosphate. These results indicate that bis-p-nitrophenyl phosphate is hydrolyzed to two moles of p-nitrophenol and one mole of inorganic phosphate without release of p-nitrophenyl phosphate as an intermediary product.
2. The kinetic parameters, Km and Vmax, for the hydrolysis of bis-p-nitrophenyl phosphate by Fusarium acid phosphatase II were determined at pH 5.3 to be 1.1mM and 0.83μmole/min/unit, respectively.
3. The kinetic parameters, Km and Vmax, for both p-nitrophenyl phosphate and bis-p-nitrophenyl phosphate showed the same pH dependence. In both cases, logVmax was independent of pH in the range pH 3.3-6.0. In the pKm-pH plot, the experimental points were on a line with a slope of -1.0 in the range pH 4.5-6.0.
4. Both activities of Fusarium acid phosphatase II for p-nitrophenyl phosphate and bis-p-nitrophenyl phosphate had the same pH optimum at 5.3 when assayed at a substrate concentration of 5mM.
5. In any mixture containing given concentrations of p-nitrophenyl phosphate and bis-p-nitrophenyl phosphate, the total velocity of the liberation of p-nitrophenol by the enzyme lay between the two velocities which were obtained with these concentrations of the substrates separately.
6. Effects of various inhibitors on both activities of the enzyme were quite similar.
7. The activity of the enzyme for bis-p-nitrophenyl phosphate was destroyed by heat faster than that for p-nitrophenyl phosphate.
8. All these results are consistent with the hypothesis that a common or at least partly common catalytic site of a single protein is responsible for hydrolyzingboth p-nitrophenyl phosphate and bis-p-nitrophenyl phosphate. A new name “phospho-diesterase-phosphomonoesterase” is proposed on the basis of enzymatic properties.
