KINETIC MECHANISM AND DIVALENT METAL ACTIVATION OF HUMAN ERYTHROCYTE PYRIDOXAL PHOSPHATASE

Human erythrocyte pyridoxal phosphatase has an essential requirement for divalent cations, Its activation by Mg2+, Co2+, Ni2+, or Mn2+ followed Michaelis-Menten kinetics, Other divalent cations inhibited the enzyme. The kinetic properties of the enzyme were investigated with pyridoxal phosphate and Mg2+ alone and in the presence of the product, P-i, or dead-end inhibitors at pH 7.4 and 37 degrees C, The enzyme bound both the substrate and Mg2+ before products were released. P-i gave competitive inhibition vs substrate and noncompetitive inhibition vs Mg2+, Molybdate also was a competitive inhibitor vs substrate and noncompetitive inhibitor vs Mg2+ Ca2+ gave competitive inhibition vs Mg2+ and noncompetitive inhibition vs substrate, The effects of Mg2+ and substrate on the inactivation of pyridoxal phosphatase by a variety of group-specific reagents were studied, The inactivation of the enzyme by iodoacetate was potentiated by MgCl2. The K-d of the enzyme-Mg complex determined in the inactivation analysis was similar to the K-m of the free enzyme for Mg2+, indicating that Mg2+ binds to the free enzyme. Low concentrations of a substrate, pyridoxine phosphate, or P-i protected pyridoxal phosphatase from inactivation by N-ethylmaleimide in the absence or presence of Mg2+. Thus, the substrate binds to the free enzyme and the enzyme-Mg complex, The steady-state kinetics and the kinetics of inactivation are consistent with random binding of pyridoxal phosphate and Mg2+ and with the formation of a dead-end complex of P-i with the enzyme-Mg complex. (C) 1995 Academic Press, Inc.