Benzophenone boronic acid photoaffinity labeling of subtilisin CMMs to probe altered specificity.
Benzophenone boronic acid photoaffinity labeling of subtilisin CMMs to probe altered specificity.
复制标题
枯草杆菌蛋白酶 CMM 的二苯甲酮硼酸光亲和标记可探测改变的特异性。
DOI：
10.1016/s0968-0896(99)00320-x
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发表时间：
2000
影响因子：
3.5
通讯作者：
J. B. Jones
中科院分区：
文献类型：
--
作者：
G. Desantis;C. Paech;J. B. Jones
利用过渡态类似物抑制剂硼酸二苯甲酮（BBP）光探针研究了化学修饰突变酶（CMMs）S1口袋拓扑结构的差异。BBP被证明是一种有效的竞争性抑制剂和显示活性位点的光探针的丝氨酸蛋白酶枯草杆菌蛋白酶迟缓芽孢杆菌（SBL）的CMMs的化学修饰的疏水性，带负电荷和带正电荷的部分在S1口袋S166 C残基。正如预期的那样，在所有情况下，BBP与WT-SBL的结合最好。与WT相比，BBP与S166 C-SCH 2C 6 H5和S166 C-CH 2-c-C6 H11（具有较大的疏水侧链）的结合分别减少了86倍和9倍。相对于WT，BBP与带电CMMs S166 C-S-CH 2CH 2SO 3-或S166 C-S-CH 2CH 2NH 3+的结合分别减少了170倍和4倍。光分解的WT-SBL-BBP酶抑制剂（EI）复合物，失活的酶，并影响WT和BBP之间的共价交联的形成。通过肽图谱分析和Edman测序在Gly 127处鉴定交联。Gly 127位于SBL的S1疏水口袋中，因此其修饰建立了S1中二苯甲酮部分的结合。在相同条件下，S166 C-SCH 2C 6 H5、S166 C-S-CH 2CH 2SO 3 −或S166 C-S-CH 2CH 2NH 3+与BBP的EI复合物的光解不会使这些酶失活，也不会影响交联的形成。这些结果证实了动力学证据，这些CMMs的活性位点拓扑结构从WT的活性位点拓扑结构显著改变。相反，虽然S166 C-CH 2-c-C6 H11-BBP EI复合物的光解在12小时后仅使50%的酶失活，但它仍然影响CMM和BBP之间共价交联的形成，再次在Gly 127处。然而，这种光解反应的效率低于WT，表明S166 C-CH 2-c-C6 H11的S1口袋与WT相比受到显著限制，但不像其他CMM那样完全。
A transition state analogue inhibitor, boronic acid benzophenone (BBP) photoprobe, was used to study the differences in the topology of the S1pocket of chemically modified mutant enzymes (CMMs). The BBP proved to be an effective competitive inhibitor and a revealing active site directed photoprobe of the CMMs of the serine protease subtilisin Bacillus lentus (SBL) which were chemically modified with the hydrophobic, negatively charged and positively charged moieties at the S1pocket S166C residue. As expected, in all cases BBP bound best to WT-SBL. BBP binding to S166C-SCH2C6H5and S166C-CH2-c-C6H11, with their large hydrophobic side chains, was reduced by 86-fold and 9-fold, respectively, compared to WT. Relative to WT, BBP binding to the charged CMMs, S166C-S-CH2CH2SO3−or S166C-S-CH2CH2NH3+, was reduced 170-fold and 4-fold respectively. Photolysis of the WT-SBL–BBP enzyme–inhibitor (EI) complex, inactivated the enzyme and effected the formation of a covalent crosslink between WT and BBP. The crosslink was identified at Gly127 by peptide mapping analysis and Edman sequencing. Gly127 is located in the S1hydrophobic pocket of SBL and its modification thus established binding of the benzophenone moiety in S1. Photolysis of the EI complex of S166C-SCH2C6H5,S166C-S-CH2CH2SO3−, or S166C-S-CH2CH2NH3+and BBP under the same conditions did not inactivate these enzymes, nor effect the formation of a crosslink. These results corroborated the kinetic evidence that the active site topology of these CMMs is dramatically altered from that of WT. In contrast, while photolysis of the S166C-CH2-c-C6H11–BBP EI complex only inactivated 50% of the enzyme after 12 h, it still effected the formation of a covalent crosslink between the CMM and BBP, again at Gly127. However, this photolytic reaction was less efficient than with WT, demonstrating that the S1pocket of S166C-CH2-c-C6H11is significantly restricted compared to WT, but not as completely as for the other CMMs.
通过用非天然氨基酸替换来检查大肠杆菌天冬氨酸转氨酶活性位点碱基 Lys-258 的结构和化学灵活性。
影响因子：
2.9
作者：
Gloss,LM;Kirsch,JF
通讯作者：
Kirsch,JF
影响因子：
2.9
作者：
DORMAN, G;PRESTWICH, GD
通讯作者：
PRESTWICH, GD
影响因子：
10.5
作者：
MACMILLAN, AM;QUERY, CC;SHARP, PA
通讯作者：
SHARP, PA