Regulation of enzymatic activity by deamidation and their subsequent repair by protein L-isoaspartyl methyl transferase.
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.
Drug Development Diagnostics, Biotechnology, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India.
Trina
T
Dutta
Shakri
S
Banerjee
Dhananjay
D
Soren
Sagar
S
Lahiri
Shinjinee
S
Sengupta
Juhi Augusta
JA
Rasquinha
Anil K
AK
Ghosh
D000577:Amides / Q000378:metabolism*; D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology; D020134:Catalytic Domain; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013696:Temperature; D014198:Trehalase / Q000737:chemistry / Q000378:metabolism*
D000577:Amides; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D014198:Trehalase; D043364:Endo-1,4-beta Xylanases