Lys515-Lys492 cross-linking by DIDS interferes with substrate utilization by the sarcoplasmic reticulum ATPase
Lys515-Lys492 cross-linking by DIDS interferes with substrate utilization by the sarcoplasmic reticulum ATPase
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DOI：
10.1016/s0006-3495(97)78245-6
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发表时间：
1997-10-01
影响因子：
3.4
通讯作者：
Inesi, G
中科院分区：
文献类型：
--
作者：
Hua, SM;Inesi, G
用4,4'-二异硫氰酸二苯乙烯-2,2'-磺酸（DIDS）衍生出肌浆网（SR） Ca2+ atp酶，并以每个atp酶一个DIDS的分子化学量产生完全的酶失活。通过多肽分析和测序确定Lys492和Lys515是由DIDS衍生的atp酶残基，在Arg505处单个色氨酸切割导致的两个肽片段缺乏电泳分辨率，表明两个衍生的残基是交联的。用DIDS交联Lys492和Lys515会干扰ATP和对硝基苯基磷酸底物的ATP酶利用，而只有Lys515与异硫氰酸荧光素衍生化会干扰ATP的ATP酶利用，但不会干扰对硝基苯基磷酸。与DIDS交联表明Lys492和Lys515之间的距离约为13埃，这与扩展构型中ATP结合的长度相对应。因此，在核苷酸结合域的凹槽内，ATP底物的腺苷部分位于Lys515附近，其末端磷酸位于Lys492附近。
Sarcoplasmic reticulum (SR) Ca2+ ATPase was derivatized with 4,4'-diisothiocyanatostilbene-2,2'-sulfonic acid (DIDS), and complete enzyme inactivation was produced with a molecular stoichiometry of one DIDS per ATPase. It was determined by peptide analysis and sequencing that Lys492 and Lys515 were the ATPase residues derivatized by DIDS, Lack of electrophoretic resolution of the two peptide fragments that result from a single tryptic cut at Arg505 demonstrated that the two derivatized residues were cross-linked. Cross-linking of Lys492 and Lys515 by DIDS interfered with ATPase utilization of both ATP and p-nitrophenyl phosphate substrates, whereas derivatization of only Lys515 with fluorescein isothiocyanate interfered with ATPase utilization of ATP but not of p-nitrophenyl phosphate. Cross-linking with DIDS implies a distance of approximately 13 Angstrom between Lys492 and Lys515, which corresponds to the length of ATP bound in an extended configuration. Therefore, within the groove of the nucleotide binding domain, the ATP substrate is positioned with the adenosine moiety near Lys515 and its terminal phosphate near Lys492.