Pyrene excimer fluorescence of yeast alcohol dehydrogenase: a sensitive probe to investigate ligand binding and unfolding pathway of the enzyme.
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. Guanidinium 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.
Santra
Manas Kumar
MK
School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai, India.
Dasgupta
Debjani
D
Panda
Dulal
D
D000426:Alcohol Dehydrogenase / Q000737:chemistry*; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D000431:Ethanol / Q000494:pharmacology; D005453:Fluorescence; D019791:Guanidine / Q000494:pharmacology; D008024:Ligands; D009243:NAD / Q000494:pharmacology; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011721:Pyrenes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D015003:Yeasts / Q000201:enzymology*; D015032:Zinc / Q000494:pharmacology
D008024:Ligands; D011721:Pyrenes; D009243:NAD; D000431:Ethanol; D014364:Tryptophan; C030984:pyrene; D000426:Alcohol Dehydrogenase; D015032:Zinc; D019791:Guanidine; D003545:Cysteine