荧光光谱法定量测定纤维素酶活性架构中单个氨基酸突变对底物结合力的影响
|
摘要
纤维素酶活性架构是酶分子中多个氨基酸残基构成的可结合并催化底物的功能区,其中色氨酸等芳香族残基在该区域中起着重要作用.本研究利用荧光光谱法,定量分析了纤维素酶Ch Cel5A活性架构中色氨酸与底物的结合动力学过程,通过色氨酸荧光猝灭的定量分析,确定了色氨酸特异性结合时的底物浓度范围,并且测定了Ch Cel5A活性架构中单个氨基酸突变导致的底物结合常数的变化,与催化动力学参数比较发现,荧光光谱法可准确表征纤维素酶与底物的结合力及其单个残基突变引起动力学参数的变化.此外,由于p NP中含有强的吸电子基团,因而以p NPC等为配体时会高估与色氨酸的结合常数约20~100倍.荧光光谱法可以测定纤维素酶结合糖分子底物的动力学参数,该方法具有灵敏和快速的特点,这为蛋白质与底物之间相互作用的定量分析提供了新的视角.
The active-site architecture of cellulase is a functional area composed of multiple amino acid residues which can bind and catalyze substrates. Aromatic residues, such as tryptophan, play important roles in the function of this region. In this study, the dynamic of binding process between tryptophan in active-site architecture of cellulase Ch Cel5 A and substrate was quantitatively analyzed using fluorescence spectrum. The substrate concentration range when tryptophan can specifically bind the substrate was determined and the change of binding constant caused by a single amino acid mutation in active-site architecture of Ch Cel5 A was measured through quantitative analysis of tryptophan fluorescence quenching. Compared with the measurement of kinetic parameters,fluorescence spectroscopy could accurately characterize the binding affinity between cellulase and substrate, as well as the change of kinetic parameter caused by a single residue mutation. Besides, when using p NPC as the ligand, the binding constant could be overestimated for 20-100 times due to the increase of strong electrophilic groups. Fluorescent spectrometry can measure the kinetic parameter of cellulase-substrate binding process in a sensitive and rapid way, providing a new perspective for the quantitatively analyzing the interaction between proteins and substrates.
The active-site architecture of cellulase is a functional area composed of multiple amino acid residues which can bind and catalyze substrates. Aromatic residues, such as tryptophan, play important roles in the function of this region. In this study, the dynamic of binding process between tryptophan in active-site architecture of cellulase Ch Cel5 A and substrate was quantitatively analyzed using fluorescence spectrum. The substrate concentration range when tryptophan can specifically bind the substrate was determined and the change of binding constant caused by a single amino acid mutation in active-site architecture of Ch Cel5 A was measured through quantitative analysis of tryptophan fluorescence quenching. Compared with the measurement of kinetic parameters,fluorescence spectroscopy could accurately characterize the binding affinity between cellulase and substrate, as well as the change of kinetic parameter caused by a single residue mutation. Besides, when using p NPC as the ligand, the binding constant could be overestimated for 20-100 times due to the increase of strong electrophilic groups. Fluorescent spectrometry can measure the kinetic parameter of cellulase-substrate binding process in a sensitive and rapid way, providing a new perspective for the quantitatively analyzing the interaction between proteins and substrates.
引文
[1]Zhang Y H P,Himmel M E,Mielenz J R.Outlook for cellulas improvement:screening and selection strategies.Biotechnolog Advances,2006,24(5):452-481
[2]顾方媛,陈朝银,石家骥,等.纤维素酶的研究进展与发展趋势微生物学杂志,2008,28(1):83-87Gu F Y,Chen Z Y,Shi J J,et al.J Microbiol,2008,28(1):83-87
[3]Ghose T K.Measurement of cellulase activities.Pure&Applied Chemistry,2009,59(2):257-268
[4]Kumar D,Murthy G S.Stochastic molecular model of enzymati hydrolysis of cellulose for ethanol production.Biotechnology fo Biofuels,2013,6(2):420-431
[5]Zhang Q,Zhang X,Wang P,et al.Determination of the action modes of cellulases from hydrolytic profiles over a time cours using fluorescence-assisted carbohydrate electrophoresis Electrophoresis,2014,36(6):910-917
[6]Himmel M E.Biomass Recalcitrance:Deconstructing the Plant Cel Wall for Bioenergy,2009
[7]Dwivedi U N,Singh P,Pandey V P,et al.Structure-function relationship among bacterial,fungal and plant laccases.Journal o Molecular Catalysis B Enzymatic,2011,68(2):117-128
[8]Nakamura A,Tsukada T,Auer S,et al.Tryptophan residue a active-site tunnel entrance of Trichoderma reesei cellobiohydrolas Cel7A is important to initiate degradation of crystalline cellulose Journal of Biological Chemistry,2013,288(19):13503-13510
[9]Zhang X,Wang S,Wu X,et al.Subsite-specific contributions o different aromatic residues in the active site architecture o glycoside hydrolase family 12.Scientific Reports,2015,5:18357
[10]Payne C M,Bomble Y J,Taylor C B,et al.Multiple functions o aromatic-carbohydrate interactions in a processive cellulas examined with molecular simulation.Journal of Biologica Chemistry,2011,286(47):41028-41035
[11]Tian L,Liu S,Wang S,et al.Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families a revealed by active-site architecture analysis.Scientific Reports2016,6:23605
[12]Lakowicz J R.Principles of fluorescence spectroscopy.Springe Berlin,2006,13(2):029901
[13]陈国珍.荧光分析法.北京:科学出版社,1990Chen G Z.The Method of Fluorescence Analysis.Beijing:Scienc Press,1990
[14]Uttam A,Chandrima J,Ravi Kiran B,et al.Exploring th mechanism of fluorescence quenching in proteins induced b tetracycline.Journal of Physical Chemistry B,2011,115(19)6312-6320
[15]Van De Weert M.Fluorescence quenching to study protein-ligand binding:common errors.Journal of Fluorescence,2010,20(2)625-629
[16]Bian Q,Liu J,Tian J,et al.Binding of genistein to human serum albumin demonstrated using tryptophan fluorescence quenching International Journal of Biological Macromolecules,2004,34(5)275-279
[17]Wang Z,Tan X,Chen D,et al.Study on the binding behavior o lysozyme with cephalosporin analogues by fluorescenc spectroscopy.Journal of Fluorescence,2009,19(5):801-808
[18]Van De Weert M,Stella L.Fluorescence quenching and ligand binding:a critical discussion of a popular methodology.Journal o Molecular Structure,2011,998(1):144-150
[19]Lombard V,Ramulu H G,Drula E,et al.The carbohydrate-activ enzymes database(CAZy)in 2013.Nucleic Acids Research,201442(D1):D490-D495
[20]Zhu Y,Zhou H,Bi Y,et al.Characterization of a family 5 glycosid hydrolase isolated from the outer membrane of cellulolyti Cytophaga hutchinsonii.Applied Microbiology and Biotechnology2013,97(9):3925-3937
[21]Medve J,Lee D,Tjerneld F.Ion-exchange chromatographi purification and quantitative analysis of Trichoderma reese cellulases cellobiohydrolaseⅠ,Ⅱand endoglucanaseⅡby fas protein liquid chromatography.Journal of Chromatography A1998,808(1-2):153-165
[22]Zhang X,Liu N,Yang F,et al.In situ demonstration and quantitative analysis of the intrinsic properties of glycosid hydrolases.Electrophoresis,2012,33(2):280-287
[23]Zhang Y H P,Cui J,Lynd L R,et al.A transition from cellulos swelling to cellulose dissolution by o-phosphoric acid:evidenc from enzymatic hydrolysis and supramolecular structure Biomacromolecules,2006,7(2):644-648
[24]Zhang L,Ma H X,Zhang H Q,et al.Thermomyces lanuginosus i the dominant fungus in maize straw composts.Bioresourc Technology,2015,197(266-275)
[25]Huang J,Xu Y,Qian X.A red-shift colorimetric and fluorescen sensor for Cu2+in aqueous solution:unsymmetrical4,5-diaminonaphthalimide with N-H deprotonation induced b metal ions.Organic&Biomolecular Chemistry,2009,7(7)1299-1303.
[26]Burstein E A,Vedenkina N S,Ivkova M N.Fluorescence and th location of tryptophan residues in protein molecules Photochemistry&Photobiology,1973,18(4):263-279
[27]Pruessner J C,Kirschbaum C,Meinlschmid G,et al.Two formula for computation of the area under the curve represent measures of total hormone concentration versus time-dependent change.Psychoneuroendocrinology,2003,28(7):916-931
[28]张勇,张贵珠,王月梅,等.光谱法研究丝裂霉素、血清白蛋白以及金属离子间的相互作用.分析科学学报,2000,16(6):445-449Zhang Y,Zhang G Z,Wang Y M,et al.Journal of Analytical Science,2000,16(6):445-449
[29]Skrt M,Benedik E,Podlipnik C,et al.Interactions of different polyphenols with bovine serum albumin using fluorescence quenching and molecular docking.Food Chemistry,2012,135(4):2418-2424
[30]边平凤.生物大分子与小分子相互作用研究.杭州:浙江大学,2008Bian P F.Study on The Interaction Between Bio-macromolecules and Small Molecules.Hangzhou:Zhejiang University,2008
[31]Palonen H,Tjerneld F,Zacchi G,et al.Adsorption of Trichoderma reesei CBHⅠand EGⅡand their catalytic domains on steam pretreated softwood and isolated lignin.Journal of Biotechnology,2004,107(1):65-72
[32]Lynd L R,Weimer P J,Van Zyl W H,et al.Microbial cellulose utilization:fundamentals and biotechnology.Microbiology&Molecular Biology Reviews,2002,66(3):739
[2]顾方媛,陈朝银,石家骥,等.纤维素酶的研究进展与发展趋势微生物学杂志,2008,28(1):83-87Gu F Y,Chen Z Y,Shi J J,et al.J Microbiol,2008,28(1):83-87
[3]Ghose T K.Measurement of cellulase activities.Pure&Applied Chemistry,2009,59(2):257-268
[4]Kumar D,Murthy G S.Stochastic molecular model of enzymati hydrolysis of cellulose for ethanol production.Biotechnology fo Biofuels,2013,6(2):420-431
[5]Zhang Q,Zhang X,Wang P,et al.Determination of the action modes of cellulases from hydrolytic profiles over a time cours using fluorescence-assisted carbohydrate electrophoresis Electrophoresis,2014,36(6):910-917
[6]Himmel M E.Biomass Recalcitrance:Deconstructing the Plant Cel Wall for Bioenergy,2009
[7]Dwivedi U N,Singh P,Pandey V P,et al.Structure-function relationship among bacterial,fungal and plant laccases.Journal o Molecular Catalysis B Enzymatic,2011,68(2):117-128
[8]Nakamura A,Tsukada T,Auer S,et al.Tryptophan residue a active-site tunnel entrance of Trichoderma reesei cellobiohydrolas Cel7A is important to initiate degradation of crystalline cellulose Journal of Biological Chemistry,2013,288(19):13503-13510
[9]Zhang X,Wang S,Wu X,et al.Subsite-specific contributions o different aromatic residues in the active site architecture o glycoside hydrolase family 12.Scientific Reports,2015,5:18357
[10]Payne C M,Bomble Y J,Taylor C B,et al.Multiple functions o aromatic-carbohydrate interactions in a processive cellulas examined with molecular simulation.Journal of Biologica Chemistry,2011,286(47):41028-41035
[11]Tian L,Liu S,Wang S,et al.Ligand-binding specificity and promiscuity of the main lignocellulolytic enzyme families a revealed by active-site architecture analysis.Scientific Reports2016,6:23605
[12]Lakowicz J R.Principles of fluorescence spectroscopy.Springe Berlin,2006,13(2):029901
[13]陈国珍.荧光分析法.北京:科学出版社,1990Chen G Z.The Method of Fluorescence Analysis.Beijing:Scienc Press,1990
[14]Uttam A,Chandrima J,Ravi Kiran B,et al.Exploring th mechanism of fluorescence quenching in proteins induced b tetracycline.Journal of Physical Chemistry B,2011,115(19)6312-6320
[15]Van De Weert M.Fluorescence quenching to study protein-ligand binding:common errors.Journal of Fluorescence,2010,20(2)625-629
[16]Bian Q,Liu J,Tian J,et al.Binding of genistein to human serum albumin demonstrated using tryptophan fluorescence quenching International Journal of Biological Macromolecules,2004,34(5)275-279
[17]Wang Z,Tan X,Chen D,et al.Study on the binding behavior o lysozyme with cephalosporin analogues by fluorescenc spectroscopy.Journal of Fluorescence,2009,19(5):801-808
[18]Van De Weert M,Stella L.Fluorescence quenching and ligand binding:a critical discussion of a popular methodology.Journal o Molecular Structure,2011,998(1):144-150
[19]Lombard V,Ramulu H G,Drula E,et al.The carbohydrate-activ enzymes database(CAZy)in 2013.Nucleic Acids Research,201442(D1):D490-D495
[20]Zhu Y,Zhou H,Bi Y,et al.Characterization of a family 5 glycosid hydrolase isolated from the outer membrane of cellulolyti Cytophaga hutchinsonii.Applied Microbiology and Biotechnology2013,97(9):3925-3937
[21]Medve J,Lee D,Tjerneld F.Ion-exchange chromatographi purification and quantitative analysis of Trichoderma reese cellulases cellobiohydrolaseⅠ,Ⅱand endoglucanaseⅡby fas protein liquid chromatography.Journal of Chromatography A1998,808(1-2):153-165
[22]Zhang X,Liu N,Yang F,et al.In situ demonstration and quantitative analysis of the intrinsic properties of glycosid hydrolases.Electrophoresis,2012,33(2):280-287
[23]Zhang Y H P,Cui J,Lynd L R,et al.A transition from cellulos swelling to cellulose dissolution by o-phosphoric acid:evidenc from enzymatic hydrolysis and supramolecular structure Biomacromolecules,2006,7(2):644-648
[24]Zhang L,Ma H X,Zhang H Q,et al.Thermomyces lanuginosus i the dominant fungus in maize straw composts.Bioresourc Technology,2015,197(266-275)
[25]Huang J,Xu Y,Qian X.A red-shift colorimetric and fluorescen sensor for Cu2+in aqueous solution:unsymmetrical4,5-diaminonaphthalimide with N-H deprotonation induced b metal ions.Organic&Biomolecular Chemistry,2009,7(7)1299-1303.
[26]Burstein E A,Vedenkina N S,Ivkova M N.Fluorescence and th location of tryptophan residues in protein molecules Photochemistry&Photobiology,1973,18(4):263-279
[27]Pruessner J C,Kirschbaum C,Meinlschmid G,et al.Two formula for computation of the area under the curve represent measures of total hormone concentration versus time-dependent change.Psychoneuroendocrinology,2003,28(7):916-931
[28]张勇,张贵珠,王月梅,等.光谱法研究丝裂霉素、血清白蛋白以及金属离子间的相互作用.分析科学学报,2000,16(6):445-449Zhang Y,Zhang G Z,Wang Y M,et al.Journal of Analytical Science,2000,16(6):445-449
[29]Skrt M,Benedik E,Podlipnik C,et al.Interactions of different polyphenols with bovine serum albumin using fluorescence quenching and molecular docking.Food Chemistry,2012,135(4):2418-2424
[30]边平凤.生物大分子与小分子相互作用研究.杭州:浙江大学,2008Bian P F.Study on The Interaction Between Bio-macromolecules and Small Molecules.Hangzhou:Zhejiang University,2008
[31]Palonen H,Tjerneld F,Zacchi G,et al.Adsorption of Trichoderma reesei CBHⅠand EGⅡand their catalytic domains on steam pretreated softwood and isolated lignin.Journal of Biotechnology,2004,107(1):65-72
[32]Lynd L R,Weimer P J,Van Zyl W H,et al.Microbial cellulose utilization:fundamentals and biotechnology.Microbiology&Molecular Biology Reviews,2002,66(3):739