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Soluble artificial metalloproteases with broad substrate selectivity, high reactivity, and high thermal and chemical stabilities

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Abstract

To design soluble artificial proteases that cleave peptide backbones of a wide range of proteins with high reactivity, artificial active sites comprising the Cu(II) complex of 1-oxa-4,7,10-triazacyclodedecane (oxacyclen) and the aldehyde group were synthesized. The aldehyde group was employed as the binding site in view of its ability to reversibly form imine bonds with ammonium groups exposed on the surfaces of proteins, and Cu(II) oxacyclen was exploited as the catalytic group for peptide hydrolysis. The artificial metalloproteases synthesized in the present study cleaved all of the protein substrates examined (albumin, γ-globulin, myoglobin, and lysozyme). In addition, the activity of the best soluble artificial protease was enhanced by up to 190-fold in terms of k cat/K m. When the temperature was raised to 80 °C, the activities of the artificial proteases were significantly enhanced. The activity of the artificial protease was not greatly affected by surfactants, including sodium dodecyl sulfate. The intermediacy of the imine complex formed between the artificial protease and the protein substrate was supported by an experiment using sodium cyanoborohydride. Soluble artificial metalloproteases with broad substrate selectivity, high reactivity, high thermal and chemical stabilities, and small molecular weights were thus synthesized by positioning the aldehyde group in proximity to Cu(II) oxacyclen.

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Abbreviations

Cyclen:

1,4,7,10-Tetraazacyclododecane

MALDI:

Matrix-assisted laser desorption/ionization

MS:

Mass spectrometry

Oxacyclen:

1-Oxa-4,7,10-triazacyclododecane

PAGE:

Polyacrylamide gel electrophoresis

SDS:

Sodium dodecyl sulfate

TOF:

Time-of-flight

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Acknowledgments

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2009-0072151). This paper is dedicated to the memory of the late Prof. Chi Sun Hahn.

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Authors and Affiliations

  1. Department of Chemistry, Seoul National University, Seoul, 151-747, Korea

    Min Gyum Kim, Sang Ho Yoo, Woo Suk Chei, Tae Yeon Lee, Hye Mi Kim & Junghun Suh

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  1. Min Gyum Kim
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  2. Sang Ho Yoo
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  3. Woo Suk Chei
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  4. Tae Yeon Lee
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  5. Hye Mi Kim
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Correspondence to Junghun Suh.

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Kim, M.G., Yoo, S.H., Chei, W.S. et al. Soluble artificial metalloproteases with broad substrate selectivity, high reactivity, and high thermal and chemical stabilities. J Biol Inorg Chem 15, 1023–1031 (2010). https://doi.org/10.1007/s00775-010-0662-x

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