Effects of Cu2+ on alkaline phosphatase from Macrobrachium rosenbergii

To gain insight into the effect of Cu 2+ on the activity and structure of alkaline phosphatase (ALP) from Macrobrachium rosenbergii , the enzyme was purified using ammonium sulfate fractionation, Sephacryl S-200, and DEAE anion exchange chromatography . We studied Cu 2+ -mediated inhibition and aggregation of ALP, and found that Cu 2+ significantly inactivated ALP activity with an IC 50 of 1.47 ± 0.02 mM. We further revealed that Cu 2+ reversibly inhibited ALP in a mixed-type manner with K i  = 0.41 ± 0.02 mM. Time-interval kinetics showed that the inhibition followed first-order reaction kinetics . This process was associated with conformational changes and significant transient free-energy change. Spectrofluorometry results showed that Cu 2+ induced ALP tertiary structural changes, including the exposure of hydrophobic surfaces that directly induced ALP aggregation. The results provide new information regarding ALP from M. rosenbergii .