大豆枯草杆菌样蛋白酶C1的裂解特异性

Patrick M. Boyd , Neel Barnaby, Anna Tan-Wilson, Karl A. Wilson
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引用次数: 29

摘要

大豆(Glycine max (L.))蛋白酶C1的裂解特异性用高效液相色谱法检测了美林(Merrill)幼苗子叶的寡肽底物。使用了一系列基于Ac-KVEKEESEEGE-NH2序列的肽,模拟了储存蛋白β-甘氨酸α亚基中蛋白酶C1的天然裂解位点。对从N端或c端截断的底物肽的研究表明,蛋白酶C2切割的最低要求是在被切割的键的N端有三个残基,c端有两个残基(即P3-P2 ')。当底物含有4到5个残基n端到被劈裂的键和4个残基c端(即P4或P5到P4 ')时,达到最大的裂解速率。通过一系列以Ac-KVEKEESEE-NH2为碱基序列的取代非肽(P5-P4 ')来检测P1, P1 '和P4位置上Glu残基的重要性。在P1位置,基于kcat/Km的相对排名是E>Q>K>A>D>F>S。P1′位置的替换得到E = Q>A>S>D>K>F,而P4′位置的替换对kcat/Km的影响较小,得到F = S = E = D>K>A = Q。这些数据表明,蛋白酶C1更倾向于在Glu-Glu和Glu-Gln键上切割,而P4 '位置的性质不太重要。寡肽的切割具有特异性,这表明蛋白酶C1对β-甘氨酸α和α′亚基的特异性较有限的切割是由于蛋白酶的序列切割特异性和底物蛋白表面适当的可剪切肽键的可及性的结合。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Cleavage specificity of the subtilisin-like protease C1 from soybean

The cleavage specificity of protease C1, isolated from soybean (Glycine max (L.) Merrill) seedling cotyledons, was examined using oligopeptide substrates in an HPLC based assay. A series of peptides based on the sequence Ac-KVEKEESEEGE-NH2 was used, mimicking a natural cleavage site of protease C1 in the α subunit of the storage protein β-conglycinin. A study of substrate peptides truncated from either the N- or C-terminus indicates that the minimal requirements for cleavage by protease C2 are three residues N-terminal to the cleaved bond, and two residues C-terminal (i.e. P3-P2′). The maximal rate of cleavage is reached with substrates containing four to five residues N-terminal to the cleaved bond and four residues C-terminal (i.e. P4 or P5 to P4′). The importance of Glu residues at the P1, P1′, and P4 positions was examined using a series of substituted nonapeptides (P5-P4′) with a base sequence of Ac-KVEKEESEE-NH2. At the P1 position, the relative ranking, based on kcat/Km, was E>Q>K>A>D>F>S. Substitutions at the P1′ position yield the ranking E≅Q>A>S>D>K>F, while those at P4′ had less effect on kcat/Km, yielding the ranking F≅S≅E≅D>K>A≅Q. These data show that protease C1 prefers to cleave at Glu-Glu and Glu-Gln bonds, and that the nature of the P4′ position is less important. The fact that there is specificity in the cleavage of the oligopeptides suggests that the more limited specific cleavage of the α and α′ subunits of β-conglycinin by protease C1 is due to a combination of the sequence cleavage specificity of the protease and the accessibility of appropriate scissile peptide bonds on the surface of the substrate protein.

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