Santra Manas Kumar, Dasgupta Debjani, Panda Dulal
School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay, Powai, Mumbai, India.
Photochem Photobiol. 2006 Mar-Apr;82(2):480-6. doi: 10.1562/2005-09-26-RA-698.
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.
酵母乙醇脱氢酶（YADH）的半胱氨酸残基被N-（1-芘基）马来酰亚胺（PM）共价修饰。每个YADH单体最多可有3.4个半胱氨酸被PM修饰。利用远紫外圆二色性发现，PM-YADH的二级结构与天然YADH的二级结构相似。PM对YADH的共价修饰抑制了酶活性，表明该酶的活性位点发生了改变。当PM与YADH每个单体亚基的掺入比例为2.6摩尔时，PM-YADH表现出最大的准分子荧光。核苷酸腺嘌呤二核苷酸（NAD）、二价锌和乙醇降低了PM-YADH的准分子荧光，表明这些试剂诱导了酶的构象变化。利用色氨酸荧光、芘准分子荧光和酶活性分析了盐酸胍（GdnHCl）诱导的YADH的去折叠。发现YADH的去折叠是逐步发生的。酶活性的丧失先于蛋白质的整体去折叠。此外，随着GdnHCl浓度增加色氨酸荧光的变化表明，2.5 M GdnHCl可使YADH完全去折叠。有趣的是，利用PM-YADH的准分子荧光，即使在存在5 M GdnHCl的情况下也检测到了YADH的残余结构。
