脂肪酶GZEL及其C-端截断突变体对磷脂单分子膜的吸附动力学研究
|
摘要
基于单分子层技术研究了禾谷镰孢菌脂肪酶GZEL对不同磷脂单分子膜的吸附动力学。同时,采用截断突变方法分析C-端肽段(编号为269-319的氨基酸)缺失后对脂肪酶GZEL界面吸附动力学参数的影响。研究发现,脂肪酶GZEL对不同磷脂单分子层的吸附动力学参数(吸附常数k_a,解离常数k_d,吸附平衡常数K_(Ads))与磷脂单分子膜的种类及初始表面压力密切相关。尽管如此,相比于野生型GZEL,269-319肽段缺失后导致酶蛋白对不同磷脂单分子膜的吸附常数k_a均显著降低,而解离常数k_d则显著升高,两者共同导致酶蛋白对于磷脂单分子膜的亲和力K_(Ads)显著降低。野生型GZEL对不同磷脂单分子膜的选择性顺序为磷脂酰丝氨酸>磷脂酰胆碱>磷脂酰乙醇胺。而269-319肽段缺失后,酶蛋白对于这三种磷脂单分子膜的亲和力则表现为无显著差异。以上结果表明269-319肽段在脂肪酶GZEL的界面吸附及对磷脂选择性方面均发挥有重要作用。
The adsorption kinetics of Gibberellazeae lipase(GZEL) onto different phospholipid monolayers was investigated based on monolayer technology. Meanwhile, was constructed to analyze the effect of deletion of C-terminal peptide(269-319 amino acids) on the adsorption kinetic parameters of GZEL onto various phospholipid monolayers was analyzed by the truncated mutation method. Results revealed that the adsorption kinetic parameters(adsorption constant k_a, dissociation constant k_d, adsorption equilibrium constant K_(Ads)) of GZEL onto different phospholipid monolayers were closely related to the type of phospholipid monolayer and initial surface pressure. Nonetheless,compared to the wild-type GZEL, a significant decrease of the adsorption constant k_a and a significant increase of the dissociation constant k_d towards different phospholipid monolayers were found after 269-319 peptide was deleted from GZEL. Both changes in adsorption and dissociation constants caused a significant reduction in the adsorption affinity(K_(Ads)) of the enzyme for various phospholipid monolayers. The order of preference for the wild-type GZEL for different phospholipid monolayers was: Phosphatidylserine > phosphatidylcholine > phosphatidylethanolamine. However, after the deletion of the 269-319 peptide, no significant difference was found in the affinity of the enzyme protein for the three phospholipid monolayers. The above results indicated that the 269-319 peptide played an important role in both the interfacial adsorption and selectivity of lipase GZEL to phospholipid monolayers.
The adsorption kinetics of Gibberellazeae lipase(GZEL) onto different phospholipid monolayers was investigated based on monolayer technology. Meanwhile, was constructed to analyze the effect of deletion of C-terminal peptide(269-319 amino acids) on the adsorption kinetic parameters of GZEL onto various phospholipid monolayers was analyzed by the truncated mutation method. Results revealed that the adsorption kinetic parameters(adsorption constant k_a, dissociation constant k_d, adsorption equilibrium constant K_(Ads)) of GZEL onto different phospholipid monolayers were closely related to the type of phospholipid monolayer and initial surface pressure. Nonetheless,compared to the wild-type GZEL, a significant decrease of the adsorption constant k_a and a significant increase of the dissociation constant k_d towards different phospholipid monolayers were found after 269-319 peptide was deleted from GZEL. Both changes in adsorption and dissociation constants caused a significant reduction in the adsorption affinity(K_(Ads)) of the enzyme for various phospholipid monolayers. The order of preference for the wild-type GZEL for different phospholipid monolayers was: Phosphatidylserine > phosphatidylcholine > phosphatidylethanolamine. However, after the deletion of the 269-319 peptide, no significant difference was found in the affinity of the enzyme protein for the three phospholipid monolayers. The above results indicated that the 269-319 peptide played an important role in both the interfacial adsorption and selectivity of lipase GZEL to phospholipid monolayers.
引文
[1]Woolley P,Steffen P.Lipases.Their Structure,Biochemistry and Application[M].Cambridge:Cambridge University Press,1994
[2]Houde A,Kademi A,Leblanc D.Lipases and their industrial applications-an overview[J].Applied Biochemistry and Biotechnology,2004,118(1-3):155-170
[3]Aarthy M,Saravanan P,Gowthaman M,et al.Enzymatic transesterification for production of biodiesel using yeast lipases:An overview[J].Chemical Engineering Research&Design,2014,92(8):1591-1601
[4]Raneva V,Ivanova T,Verger R,et al.Comparative kinetics of phospholipase A2 action on liposomes and monolayers of phosphatidylcholine spread at the air-water interface[J].Colloids and Surfaces B:Biointerfaces,1995,3(6):357-369
[5]Verger R.Interfacial activation of lipases:facts and artifacts[J].Trends in Biotechnology,1997,15(1):32-38
[6]Momsen W,Brockman H.The adsorption to and hydrolysis of 1,3-didecanoyl glycerol monolayers by pancreatic lipase.Effects of substrate packing density[J].Journal of Biological Chemistry,1981,256(13):6913-6916
[7]Ransac S,Moreau H,Riviere C,et al.Monolayer techniques for studying phospholipase kinetics[J].Methods in Enzymology,1991,197:49-65
[8]Brezesinski G,M?hwald H.Langmuir monolayers to study interactions at model membrane surfaces[J].Advances in Colloid&Interface Science,2003,100(2):563-584
[9]Brockman H.Lipid monolayers:why use half a membrane to characterize protein-membrane interactions[J].Current Opinion in Structural Biology,1999,9(4):438-443
[10]Boucher J,Trudel E,Méthot M,et al.Organization,structure and activity of proteins in monolayers[J].Colloids&Surfaces B Biointerfaces,2007,58(2):73-90
[11]Magetdana R.The monolayer technique:a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes[J].Biochimica Biophysica Acta(BBA)-Biomembranes,1999,1462(1-2):109-140
[12]Boisselieré,Demersé,Cantin L,et al.How to gather useful and valuable information from protein binding measurements using Langmuir lipid monolayers[J].Advances in Colloid and Interface Science,2017,243:60-76
[13]Phillips M,Sparks C.properties of apolipoprotens at the air-water interface[J].Annals of the New York Academy of Sciences,1980,348(1):122-137
[14]Bénarouche A,Point V,Parsiegla G,et al.New insights into the pH-dependent interfacial adsorption of dog gastric lipase using the monolayer technique[J].Colloids and Surfaces B:Biointerfaces,2013,111:306-312
[15]Desjardins A,Proctor H,Bairoch A,et al.Reduced virulence of trichothecene nonproducing mutants of gibberellazeae in wheat field tests[J].Mol.Plant Microbe Interact,1996,9:775-781
[16]Mcmullen M,Jones R,Gallenberg D.Scab of wheat and barley:A re-emerging disease of devasting impact[J].Plant Disease,1997,81(12):1340-1348
[17]Nganje W,Bangsund D,Leistritz F,et al.Regional economic impacts of Fusarium head blight in wheat and barley[J].Review of Agricultural Economics,2004,26(3):332-347
[18]Lou Z,Li M,Sun Y,et al.Crystal structure of a secreted lipase from gibberellazeae reveals a novel“double-lock”mechanism[J].Protein&Cell,2010,1(8):760-770
[19]Wang F,Zhang H,Zhao Z,et al.Recombinant lipase from gibberellazeae exhibits broad substrate specificity:Acomparative study on emulsified and monomolecular substrate[J].International Journal of Molecular Sciences,2017,18(7):1535
[2]Houde A,Kademi A,Leblanc D.Lipases and their industrial applications-an overview[J].Applied Biochemistry and Biotechnology,2004,118(1-3):155-170
[3]Aarthy M,Saravanan P,Gowthaman M,et al.Enzymatic transesterification for production of biodiesel using yeast lipases:An overview[J].Chemical Engineering Research&Design,2014,92(8):1591-1601
[4]Raneva V,Ivanova T,Verger R,et al.Comparative kinetics of phospholipase A2 action on liposomes and monolayers of phosphatidylcholine spread at the air-water interface[J].Colloids and Surfaces B:Biointerfaces,1995,3(6):357-369
[5]Verger R.Interfacial activation of lipases:facts and artifacts[J].Trends in Biotechnology,1997,15(1):32-38
[6]Momsen W,Brockman H.The adsorption to and hydrolysis of 1,3-didecanoyl glycerol monolayers by pancreatic lipase.Effects of substrate packing density[J].Journal of Biological Chemistry,1981,256(13):6913-6916
[7]Ransac S,Moreau H,Riviere C,et al.Monolayer techniques for studying phospholipase kinetics[J].Methods in Enzymology,1991,197:49-65
[8]Brezesinski G,M?hwald H.Langmuir monolayers to study interactions at model membrane surfaces[J].Advances in Colloid&Interface Science,2003,100(2):563-584
[9]Brockman H.Lipid monolayers:why use half a membrane to characterize protein-membrane interactions[J].Current Opinion in Structural Biology,1999,9(4):438-443
[10]Boucher J,Trudel E,Méthot M,et al.Organization,structure and activity of proteins in monolayers[J].Colloids&Surfaces B Biointerfaces,2007,58(2):73-90
[11]Magetdana R.The monolayer technique:a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes[J].Biochimica Biophysica Acta(BBA)-Biomembranes,1999,1462(1-2):109-140
[12]Boisselieré,Demersé,Cantin L,et al.How to gather useful and valuable information from protein binding measurements using Langmuir lipid monolayers[J].Advances in Colloid and Interface Science,2017,243:60-76
[13]Phillips M,Sparks C.properties of apolipoprotens at the air-water interface[J].Annals of the New York Academy of Sciences,1980,348(1):122-137
[14]Bénarouche A,Point V,Parsiegla G,et al.New insights into the pH-dependent interfacial adsorption of dog gastric lipase using the monolayer technique[J].Colloids and Surfaces B:Biointerfaces,2013,111:306-312
[15]Desjardins A,Proctor H,Bairoch A,et al.Reduced virulence of trichothecene nonproducing mutants of gibberellazeae in wheat field tests[J].Mol.Plant Microbe Interact,1996,9:775-781
[16]Mcmullen M,Jones R,Gallenberg D.Scab of wheat and barley:A re-emerging disease of devasting impact[J].Plant Disease,1997,81(12):1340-1348
[17]Nganje W,Bangsund D,Leistritz F,et al.Regional economic impacts of Fusarium head blight in wheat and barley[J].Review of Agricultural Economics,2004,26(3):332-347
[18]Lou Z,Li M,Sun Y,et al.Crystal structure of a secreted lipase from gibberellazeae reveals a novel“double-lock”mechanism[J].Protein&Cell,2010,1(8):760-770
[19]Wang F,Zhang H,Zhao Z,et al.Recombinant lipase from gibberellazeae exhibits broad substrate specificity:Acomparative study on emulsified and monomolecular substrate[J].International Journal of Molecular Sciences,2017,18(7):1535