Effects of multivalency and hydrophobicity of polyamines on enzyme hyperactivation of α-chymotrypsin

• Amine compounds as additive enhanced the enzyme activity of α-chymotrypsin (ChT).
• Multivalency and hydrophobicity of the amine compounds enhances of ChT activity.
• Enhancement of the amine compound was caused by catalytic constant (kcat), rather than Michaelis constant (KM).
• The increased kcat results from the interaction between ChT and amine compounds.

Enzyme hyperactivation using a complementary charged modulator/substrate pair has potential for enzyme applications in a wide range of research fields. Here, we report that amine compounds as well as naturally occurring polyamines, putrescine, spermidine, and spermine, enhance the enzyme activity of α-chymotrypsin (ChT) toward anionic substrates. The enzyme activity of ChT was increased by 1.6–6.9-fold in the presence of 50 mM amine compounds at pH 7.5. Analysis of the enzyme kinetics indicated that the catalytic constant (kcat) and specific constant (kcat/KM) increased in the presence of amine compounds depending on both the multivalency and hydrophobicity of the amine compounds, whereas the Michaelis constant (KM) remained constant. Molecular dynamics simulation suggested that the enhancement of enzyme activity of ChT was induced by weak interactions between amine compounds and ChT. These results provide extended design parameters for artificial activators for enzyme hyperactivation as well as an understanding of enzyme activity in the crude complex condition in vivo.

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