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, Pi, or dead-end inhibitors at pH 7.4 and 37 degrees C. The enzyme bound both the substrate and Mg2+ before products were released. Pi 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 Kd of the enzyme-Mg complex determined in the inactivation analysis was similar to the Km of the free enzyme for Mg2+, indicating that Mg2+ binds to the free enzyme. Low concentrations of a substrate, pyridoxine phosphate, or Pi 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 Pi with the enzyme-Mg complex.
Fonda
M L
ML
Department of Biochemistry, University of Louisville School of Medicine, Kentucky 40292, USA.
Zhang
Y N
YN
D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*
D008670:Metals; C022493:pyridoxal-5-phosphatase; D010744:Phosphoric Monoester Hydrolases