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Enzyme Kinetics Features of the Representative Engineered Recombinants of Chondroitinase ABC I

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Abstract

Chondroitinase ABC I (cABC I) from Proteus vulgaris is an important enzyme in medicinal biotechnology due to its ability to help axon regeneration after spinal cord injury. Its practical application involves solving several problems at the molecular and cellular levels. Structurally, most residues at the C-terminal domain of cABC I are arranged as organized strands, and only a small fraction of residues have helical conformation. The structural and functional features of modified residues on two specific helix fragments have previously been reported. The single mutant M889K has been combined with L679S and L679D mutants to make enzyme variants containing simultaneously modified helix. Here, the pH stability and temperature-based analysis of the transition state structure for the catalysis reaction were investigated. We found that double mutant L679D/M889K is the better choice to use in physiological conditions due to its higher pH stability at physiological pH as well as its different optimum temperature as compared with the (wild-type) WT protein. According to Arrhenius’s analysis, the values of the Gibbs free energy of the transition state (∆G#) are not changed upon mutation. However, the relative contribution and absolute values of the enthalpy and entropy change to the total value of ∆G#, varied between the WT and mutants.

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Acknowledgements

We appreciate the Tarbiat Modares University of Tehran for the technical support of the research. The authors have declared no conflict of interest. Dr. Khosro Khajeh and Dr. Abolfazl Golestani are acknowledged for their generous donation of bacterium.

Funding

This work was supported by the research council of the University of Zanjan.

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Authors and Affiliations

  1. Department of Biology, Faculty of Sciences, University of Zanjan, Zanjan, Iran

    Khadijeh Moradi, Zahra Bayani, Vahab Jafarian & Akram Shirdel

  2. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

    Akram Shirdel

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  1. Khadijeh Moradi
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  2. Zahra Bayani
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Correspondence to Vahab Jafarian or Akram Shirdel.

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10930_2023_10093_MOESM1_ESM.tif

Supplementary file1 (TIF 90 KB)Supplementary image 1. SDS-PAGE analysis of purified recombinant proteins. Lane 1, marker; lane2, WT; lane3, M889K; lane 4, M889L; lane 5, L679D/M889K; lane 6, L679S/M889K. The numbers provided on the left side of the image are the molecular weight of marker proteins in kDa.

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Moradi, K., Bayani, Z., Jafarian, V. et al. Enzyme Kinetics Features of the Representative Engineered Recombinants of Chondroitinase ABC I. Protein J 42, 55–63 (2023). https://doi.org/10.1007/s10930-023-10093-w

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