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Toward the engineering of a super efficient enzyme.

S Folcarelli1, F Venerini, A Battistoni

  • 1Department of Biology, University of Rome "Tor Vergata,", Rome, 00133, Italy.

Biochemical and Biophysical Research Communications
|March 18, 1999
PubMed
Summary

A mutant superoxide dismutase enzyme exhibits record-breaking catalytic activity. This enhanced efficiency, driven by improved substrate attraction and active-site accessibility, approaches the theoretical diffusion limit for enzymes.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Engineering

Background:

  • Superoxide dismutase (SOD) enzymes are crucial for cellular defense against reactive oxygen species.
  • The catalytic efficiency of SODs is vital for their biological function.
  • Understanding structure-activity relationships in SODs can lead to improved enzyme variants.

Purpose of the Study:

  • To investigate the catalytic activity of a mutant Photobacterium leiognathi Cu, Zn superoxide dismutase (SOD) with a Glu59Gln substitution.
  • To elucidate the factors contributing to the enzyme's enhanced catalytic performance.

Main Methods:

  • Pulse radiolysis was used to measure the catalytic activity (kcat/KM) of the mutant enzyme.
  • Brownian dynamics simulations were employed to analyze substrate attraction and electric field distribution.

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  • Iodide interaction studies were conducted to assess active-site accessibility.

    • Main Results:

    • The Glu59Gln mutant exhibited a kcat/KM of 1.0 x 10(10) M-1s-1, the highest recorded for any SOD.
    • Simulations indicated enhanced substrate attraction due to altered electric field distribution.
    • The mutant's active site showed increased solvent accessibility, with iodide binding twice as strongly as in the native enzyme.

    Conclusions:

    • The P. leiognathi Cu, Zn SOD Glu59Gln mutant represents the most efficient enzyme characterized to date, approaching the diffusion limit.
    • Modified electric field distribution and increased active-site solvent accessibility are key factors in its superior performance.
    • This engineered SOD demonstrates the potential for optimizing enzyme activity through targeted mutagenesis.