Effects of ATP and CTP on the conformation of the regulatory subunit of Escherichia coli aspartate transcarbamylase in solution: a medium-resolution hydrogen exchange study.
Effects of ATP and CTP on the conformation of the regulatory subunit of Escherichia coli aspartate transcarbamylase in solution: a medium-resolution hydrogen exchange study.
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DOI：
10.1021/bi00439a011
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发表时间：
1989-06
期刊：
影响因子：
2.9
通讯作者：
D. Mallikarachchi;D. Burz;N. Allewell
中科院分区：
文献类型：
--
作者：
D. Mallikarachchi;D. Burz;N. Allewell
中分辨率氢交换方法已被用来检查在存在和不存在ATP和CTP的情况下，在大肠杆菌天冬氨酸转氨甲酰酶的调节亚基（R2）的7个肽的溶剂可及性。两种核苷酸都是全酶的变构效应物; ATP的结合增加全酶对底物L-Asp的亲和力，而CTP具有相反的作用。根据Rosa和理查兹（1979，1981，1982）以及Englander等人（1983，1985）的研究，通过将pH值调节至2.7以淬灭交换，用胃蛋白酶消化蛋白质，通过反相HPLC分离肽，测定其放射性，并校正分析期间的放射性损失，生成单个肽的交换曲线。通过氨基酸分析和N-末端分析鉴定了来自片段1-11和76-153的16个肽。其中9个区域的背景浓度过高或浓度过低，无法监测交换情况。在肽1-11、76-91、78-90、84-101、93-112、108-114和115-125中可遵循其交换的质子数范围为约1（1-11、108-114）至10（84-101）和11（93-112）。所获得的结果的模式表明，在溶液中的r2的结构是类似的晶体ATCase中的调节亚基。CTP和ATP都降低了除115-125之外的所有七种肽的交换速率。尽管CTP比ATP更能减缓交换，但除了靠近核苷酸结合位点的肽76-91和78-90外，其影响很小。（250字处删节）
Medium-resolution hydrogen exchange methods have been used to examine the solvent accessibility of seven peptides in the regulatory subunit (r2) of Escherichia coli aspartate transcarbamylase in the presence and absence of ATP and CTP. Both nucleotides are allosteric effectors of the holoenzyme; binding of ATP increases the affinity of the holoenzyme for the substrate L-Asp, while CTP has the opposite effect. Following Rosa and Richards (1979, 1981, 1982) and Englander et al. (1983, 1985), exchange-out curves for individual peptides were generated by adjusting the pH to 2.7 to quench exchange-out, digesting the protein with pepsin, separating peptides by reverse-phase HPLC, determining their radioactivity, and correcting for radioactivity lost during the analysis. Sixteen peptides from segments 1-11 and 76-153 were identified by amino acid and N-terminal analysis. Nine fell in regions where background was too high or were present at too low concentrations for exchange to be monitored. The number of protons whose exchange could be followed in peptides 1-11, 76-91, 78-90, 84-101, 93-112, 108-114, and 115-125 ranged from approximately 1 (1-11, 108-114) to 10 (84-101) and 11 (93-112). The pattern of results obtained suggests that the structure of r2 in solution is similar to that of the regulatory subunits in crystalline ATCase. Both CTP and ATP reduce rates of exchange from all seven peptides except 115-125. Although CTP slows exchange more than ATP, the effect is small except for peptides 76-91 and 78-90 which are near the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)