Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates
Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates
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DOI：
10.1074/jbc.m008702200
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发表时间：
2001-01-12
影响因子：
4.8
通讯作者：
Hersh, LB
中科院分区：
文献类型：
--
作者：
Song, ES;Mukherjee, A;Hersh, LB
重组大鼠胰岛素被证明能在R-K键上裂解内部淬灭的荧光肽2-氨基苄基-GGFLRKVGQ-乙二胺-2，4-二硝基苯酚，表现出K-m为13 μ M和V-max为2.6 μ mol min（-1）mg（-1）。该肽的P-2亮氨酸或P-2'缬氨酸被其他残基取代的衍生物用于探测酶的亚位点特异性。改变P-2残基产生K-m的1倍范围和k（cat）的7倍范围。P-2残基的性质对切割位点有显著影响。亮氨酸、异亮氨酸、缬氨酸和天冬氨酸在R-K键产生裂解，天冬酰胺在N-R键产生36%裂解，在RK键产生64%裂解，而丙氨酸或丝氨酸的A-R和S-R键是主要裂解位点。用酪氨酸、苯丙氨酸、甲硫氨酸或组氨酸代表不同的残基X，在F-X、X-R和RK处观察到裂解，而用色氨酸，在F-W和W-R键处发生相等的裂解。可变的P-2'残基在K-m和k（cat）两者中产生较小的变化，并且对切割位点几乎没有影响。它们是苯丙氨酸、酪氨酸、亮氨酸和异亮氨酸，它们除了在RK键处产生裂解外，还在GR键处产生显著裂解。丙氨酸和酪氨酸在F-L键处产生断裂是独特的。两者合计，这些数据表明，胰岛素特异性是针对疏水性和碱性残基的氨基侧，该酶有一个扩展的底物结合位点。
Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K-m of 13 muM and a V-max of 2.6 mu mol min(-1) mg(-1). Derivatives of this peptide in which the P-2 leucine or the P-2' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P-2 residue produced a l-fold range in K-m and a 7-fold range in k(cat). The nature of the P-2 residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond, Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the RK bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and RK were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P-2' residues produce less of a change in both K-m and k(cat) and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the RK bond, produce significant cleavage at the GR bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.