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Pyrene Excimer Fluorescence of Yeast Alcohol Dehydrogenase: A Sensitive Probe to Investigate Ligand Binding and Unfolding Pathway of the Enzyme
Author(s) -
Santra Manas Kumar,
Dasgupta Debjani,
Panda Dulal
Publication year - 2006
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1562/2005-09-26-ra-698
Subject(s) - chemistry , excimer , pyrene , fluorescence , monomer , alcohol dehydrogenase , fluorescence spectrometry , tryptophan , enzyme , biochemistry , organic chemistry , physics , quantum mechanics , polymer , amino acid
The cysteine residues of yeast alcohol dehydrogenase (YADH) were covalently modified by N‐(1‐pyrenyl) maleimide (PM). A maximum of 3.4 cysteines per YADH monomer could be modified by PM. The secondary structure of PM‐YADH was found to be similar to that of the native YADH using far‐UV circular dichroism. The covalent modification of YADH by PM inhibited the enzymatic activity indicating that the active site of the enzyme was altered. PM‐YADH displayed maximum excimer fluorescence at an incorporation ratio of 2.6 mol of PM per monomeric subunit of YADH. Nucleotide adenine dinucleotide (NAD) divalent zinc and ethanol reduced the excimer fluorescence of PM‐YADH indicating that these agents induce conformational changes in the enzyme. Guani‐dinium hydrochloride (GdnHCl)‐induced unfolding of YADH was analyzed using tryptophan fluorescence, pyrene excimer fluorescence and enzymatic activity. The unfolding of YADH was found to occur in a stepwise manner. The loss of enzymatic activity preceded the global unfolding of the protein. Further, changes in tryptophan fluorescence with increasing GdnHCl suggested that YADH was completely unfolded by 2.5 M GdnHCl. Interestingly, residual structures of YADH were detected even in the presence of 5 M GdnHCl using the excimer fluorescence of PM‐YADH.