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Regulation of Enzymatic Activity by Deamidation and Their Subsequent Repair by Protein l-isoaspartyl Methyl Transferase

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Abstract

The present study explored both spontaneous and stress-induced deamidation in acid trehalase and endo-xylanase. An alteration in optimum pH by 1.5 units and optimum temperature by 20 °C accelerated the process of deamidation with a rise in isoaspartate formation and ammonia loss. Spontaneous deamidation during an enzyme-substrate reaction at physiological conditions resulted in accretion of isoaspartyl residues within the enzymes which gradually impaired their catalytic efficacy. Deamidation appeared to be more pronounced in endo-xylanase owing to its secondary structure conformation and high asparagine content. The active sites, Ala 549 in acid trehalase and His184 and Trp188 in endo-xylanase contributed to the loss of enzyme activity as they were flanking the deamidation-susceptible Asn residues. Protein l-isoaspartyl methyl transferase seemed to have a repairing capability, which enabled the heat-damaged enzymes to regain their partial activity as evident from there rise in K cat/K m. Endo-xylanase could regain 38.1 % of its biological activity while a lesser 17.5 % reactivation was obtained in acid trehalase. A unique protein l-isoaspartyl methyl transferase recognition site, Asn 151 was also identified in acid trehalase. A mass increment of the tryptic peptides of repaired enzyme due to methylation catalyzed by protein l-isoaspartyl methyl transferase substantiated the repair hypothesis.

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Abbreviations

EX:

Endo-xylanase

AT:

Acid trehalase

ADH:

Alcohol dehydrogenase

Asn:

Asparagine

PIMT:

Protein l-isoaspartyl methyl transferase

IsoAsp:

Isoaspartate

l-Asp:

l-aspartate

ES complex:

Enzyme-substrate complex

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Acknowledgments

Authors wish to thank The Director, IICB, for financial support from CSIR, Network Project NWP 0005 and Mr. Sandeep Chakraborty for his assistance in MALDI-TOF analysis.

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Authors and Affiliations

  1. Drug Development Diagnostics, Biotechnology, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata, 700032, India

    Trina Dutta, Shakri Banerjee, Dhananjay Soren, Sagar Lahiri, Shinjinee Sengupta, Juhi Augusta Rasquinha & Anil K. Ghosh

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  1. Trina Dutta
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  2. Shakri Banerjee
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  3. Dhananjay Soren
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  7. Anil K. Ghosh
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Correspondence to Anil K. Ghosh.

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Dutta, T., Banerjee, S., Soren, D. et al. Regulation of Enzymatic Activity by Deamidation and Their Subsequent Repair by Protein l-isoaspartyl Methyl Transferase. Appl Biochem Biotechnol 168, 2358–2375 (2012). https://doi.org/10.1007/s12010-012-9942-y

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