The hydrophobicity of an amino acid residue in a flexible loop of KP-43 protease alters activity toward a macromolecule substrate.

ABSTRACT

KP-43, a 43-kDa alkaline serine protease, is resistant to chemical oxidants and surfactants, making it suitable for use in laundry detergents. An amino acid residue at position 195, in a unique flexible loop that binds a Ca2+ ion, dramatically affects the proteolytic activity and thermal stability of KP-43. In the present study, we obtained 20 variants with substitutions at position 195 and investigated how these residues affect hydrolytic activity toward a macromolecular substrate (casein) and a synthetic tetra-peptide (AAPL). At pH 10, the variant with the highest caseinolytic activity, Tyr195Gln, exhibited 4.4-fold higher activity than the variant with the lowest caseinolytic activity, Tyr195Trp. A significant negative correlation was observed between the hydrophobicity of the residue at position 195 and caseinolytic activity at pH 8-10. At pH 7, the correlation became weak; at pH 6, the correlation reversed to positive. Unlike casein, in the case of hydrolysis of AAPL, no correlation was observed at pH 10 or pH 6. Because the amino acid residue at position 195 is located on the protein surface and considered sufficiently far from the active cleft, the variation in caseinolytic activity between the 20 variants was attributed to changes in interaction efficiency with different states of casein at different pH values. To improve the enzymatic activity, we propose substituting amino acid residues on the protein surface to change the efficiency of interaction with the macromolecular substrates. KEY POINTS • A single amino acid residue on the protein surface markedly changed enzyme activity. • The hydrophobicity of the amino acid residue and enzyme activity had a correlation. • The key amino acid residue for substrate recognition exists on the protein surface.