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首页> 外文期刊>Biochemistry >Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin a (stefin a) substantially enhances inhibition of cysteine proteinases.
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Grafting of features of cystatins C or B into the N-terminal region or second binding loop of cystatin a (stefin a) substantially enhances inhibition of cysteine proteinases.

机译：将半胱氨酸蛋白酶抑制剂C或B的特征接枝到半胱氨酸蛋白酶抑制剂a（Stefin a）的N末端区域或第二结合环中，实质上增强了对半胱氨酸蛋白酶的抑制。

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摘要

Replacement of the three N-terminal residues preceding the conserved Gly of cystatin A by the corresponding 10-residue long segment of cystatin C increased the affinity of the inhibitor for the major lysosomal cysteine proteinase, cathepsin B, by approximately 15-fold. This tighter binding was predominantly due to a higher overall association rate constant. Characterization of the interaction with an inactive Cys29 to Ala variant of cathepsin B indicated that the higher rate constant was a result of an increased ability of the N-terminal region of the chimeric inhibitor to promote displacement of the cathepsin B occluding loop in the second binding step. The low dissociation rate constant for the binding of cystatin A to cathepsin B was retained by the chimeric inhibitor, which therefore had a higher affinity for this enzyme than any natural cystatin identified so far. In contrast, the N-terminal substitution negligibly affected the ability of cystatin A to inhibit papain. However, substitutions of Gly75 in the second binding loop of cystatin A by Trp or His, making the loop similar to those of cystatins C or B, respectively, increased the affinity for papain by approximately 10-fold. This enhanced affinity was due to both a higher association rate constant and a lower dissociation rate constant. Modeling of complexes between the two variants and papain indicated the possibility of favorable interactions being established between the substituting residues and the enzyme. The second-loop substitutions negligibly affected or moderately reduced the affinity for cathepsin B. Together, these results show that the inhibitory ability of cystatins can be substantially improved by protein engineering.
机译：胱抑素C的相应10个残基长片段取代了胱抑素A保守Gly之前的三个N末端残基，使抑制剂对主要溶酶体半胱氨酸蛋白酶组织蛋白酶B的亲和力增加了约15倍。这种更紧密的结合主要是由于较高的总缔合速率常数。与组织蛋白酶B的非活性Cys29到Ala变体相互作用的特征表明，较高的速率常数是由于嵌合抑制剂N端区域增强组织蛋白酶B封闭环在第二次结合中的置换能力的结果步。嵌合抑制剂保留了半胱氨酸蛋白酶抑制剂A与组织蛋白酶B结合的低解离速率常数，因此与迄今为止确定的任何天然半胱氨酸蛋白酶抑制剂相比，该抑制剂对该酶的亲和力更高。相反，N-末端取代对半胱氨酸蛋白酶抑制剂A抑制木瓜蛋白酶的影响可忽略不计。但是，在胱抑素A的第二个结合环中被Trp或His取代Gly75，使该环分别类似于胱抑素C或B的环，使对木瓜蛋白酶的亲和力提高了约10倍。这种增强的亲和力归因于较高的缔合速率常数和较低的解离速率常数。对两个变体和木瓜蛋白酶之间的复合物进行建模表明，在取代残基和酶之间建立有利相互作用的可能性。第二环取代可忽略不计地影响或适度降低了对组织蛋白酶B的亲和力。总而言之，这些结果表明，通过蛋白质工程可以大大提高半胱氨酸蛋白酶抑制剂的抑制能力。

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