KINETIC MECHANISM AND DIVALENT METAL ACTIVATION OF HUMAN ERYTHROCYTE PYRIDOXAL PHOSPHATASE

Human erythrocyte pyridoxal phosphatase has an essential requirement f or divalent cations, Its activation by Mg2+, Co2+, Ni2+, or Mn2+ follo wed Michaelis-Menten kinetics, Other divalent cations inhibited the en zyme. The kinetic properties of the enzyme were investigated with pyri doxal 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 co mpetitive inhibition vs substrate and noncompetitive inhibition vs Mg2 +, Molybdate also was a competitive inhibitor vs substrate and noncomp etitive inhibitor vs Mg2+ Ca2+ gave competitive inhibition vs Mg2+ and noncompetitive inhibition vs substrate, The effects of Mg2+ and subst rate on the inactivation of pyridoxal phosphatase by a variety of grou p-specific reagents were studied, The inactivation of the enzyme by io doacetate was potentiated by MgCl2. The K-d of the enzyme-Mg complex d etermined in the inactivation analysis was similar to the K-m of the f ree enzyme for Mg2+, indicating that Mg2+ binds to the free enzyme. Lo w concentrations of a substrate, pyridoxine phosphate, or P-i protecte d pyridoxal phosphatase from inactivation by N-ethylmaleimide in the a bsence or presence of Mg2+. Thus, the substrate binds to the free enzy me and the enzyme-Mg complex, The steady-state kinetics and the kineti cs of inactivation are consistent with random binding of pyridoxal pho sphate and Mg2+ and with the formation of a dead-end complex of P-i wi th the enzyme-Mg complex. (C) 1995 Academic Press, Inc.