Noncovalent binding of polyethylene glycol (PEG) to a protein surface is a unique protein handling technique to control protein function and stability. A diblock copolymer containing PEG and polyelectrolyte chains (PEGylated polyelectrolyte) is a promising candidate for noncovalent attachment of PEG to a protein surface because of the binding through multiple electrostatic interactions without protein denaturation. To obtain a deeper understanding of protein polyelectrolyte interaction at the molecular level, we investigated the manner in which cationic PEGylated poly electrolyte binds to anionic alpha-amylase in enzyrrie kinetic experiments and molecular dynamics (MD) simulations. Cationic PEG-block-poly(N,N-dimethylarninoethyl) (PEG-b-PAMA) inhibited, the enzyme activity of anionic alpha-amylase due to binding of PAMA chains. Enzyme kinetics revealed that the inhibition of alpha-amylase activity by PEG-b-PAMA is noncompetitive inhibition manner. In MD simulations, the PEG-b-PAMA molecule was initially located at six different placements of the x-, y-, and z-axis +/- 20 angstrom from the center of alpha-amylase, which Showed that the PEG-b-PAMA nonspecifically bound to the alpha-amylase surface, corresponding to the noncompetitive inhibition manner that stems from the polymer binding to an enzyme surface other than the active site. In addition, the enzyme activity of a-amylase in the presence of PEG-b-PAMA was not inhibited by increasing the ionic strength, consistent With the MD simulation; i.e., PEG-b-PAMA did not interact with a amylase in high ionic strength conditions. The results reported in this paper suggest that enzyme inhibition by PEGylated polyelectrolyte can be attributed to the random electrostatic interaction between protein and polyelectrolyte.