CRYSTAL-STRUCTURES OF PHOSPHONOACETAMIDE LIGATED-T AND PHOSPHONOACETAMIDE AND MALONATE LIGATED-R STATES OF ASPARTATE CARBAMOYLTRANSFERASE AT 2.8-A RESOLUTION AND NEUTRAL PH
CRYSTAL-STRUCTURES OF PHOSPHONOACETAMIDE LIGATED-T AND PHOSPHONOACETAMIDE AND MALONATE LIGATED-R STATES OF ASPARTATE CARBAMOYLTRANSFERASE AT 2.8-A RESOLUTION AND NEUTRAL PH
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DOI：
10.1021/bi00454a013
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发表时间：
1990-01-16
期刊：
影响因子：
2.9
通讯作者：
LIPSCOMB, WN
中科院分区：
文献类型：
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作者：
GOUAUX, JE;LIPSCOMB, WN
T.fwdarw。合作酶天冬氨酸氨基甲酰转移酶的R转变发生在单晶中的pH为7时，没有明显的破裂，使那些未破裂的晶体适合于单晶X射线分析。来宣传T.fwdarw。R转换时，我们使用了氨基甲酰磷酸和天冬氨酸的竞争性抑制剂，它们分别是磷酰乙酰胺(PAM)和丙二酸。作为对PAM与T-态晶体结合的反应，残基Thr 53-Thr 55和Pro 266-Pro 268移动到它们的R-态位置，与PAM的膦酸基和氨基结合。这些变化诱导了一种构象，可以紧密结合天冬氨酸类似物丙二酸，从而影响变构转变。我们证明了这一点，当在pH值为7的含有20 mM PAM和8 mM丙二酸盐的溶液中转移到含有20 mM PAM和8 mM丙二酸盐的溶液中时，PAM连接的T态晶体(TPAM)，空间群P321(a=122.2，c=142.2)，异构化为R态晶体(RPAM，MAL，浸泡)，空间群P321(a=122.2，c=156.4)。T.fwdarw的R状态结构。在pH值为7的晶体内发生的R转变与在pH 7下确定的R态结构(所有原子的均方根=0.19)非常好地匹配，在该结构中，晶体最初在pH为5.9的PAM和丙二酸盐的溶液中生长，然后转移到含有pH为7的配体的缓冲液中(Rpam，Mal，Crys)。事实上，PAM和丙二酸连接的R-态结构都非常类似于氨基甲酰磷酸和琥珀酸或N-(膦-乙酰基)-L-天冬氨酸连接的结构，尽管这里报道的R-态结构是在pH 7下测定的。三种结构的结晶残留物在2.8-.ANG.分辨率下被细化到0.16-0.18。
The T .fwdarw. R transition of the cooperative enzyme aspartate carbamoyltransferase occurs at pH 7 in single crystals without visibly cracking many of the crystals and leaving those uncracked suitable for single-crystal X-ray analysis. To promote the T .fwdarw. R transition, we employ the competitive inhibitors of carbamoyl phosphate and aspartate, which are phosphonoacetamide (PAM) and malonate, respectively. In response to PAM binding to the T-state crystals, residues Thr 53-Thr 55 and Pro 266-Pro 268 move to their R-state positions to bind to the phosphonate and amino group of PAM. These changes induce a conformation that can bind tightly the aspartate analogue malonate, which thereby effects the allosteric transition. We prove this by showing that PAM-ligated T-state crystals (Tpam), space group P321 (a = 122.2 .ANG., c = 142.2 .ANG.), when transferred to a solution containing 20 mM PAM and 8 mM malonate at pH 7, isomerize to R-state crystals (Rpam,mal,soak), space group also P321 (a = 122.2 .ANG., c = 156.4 .ANG.). The R-state structure in which the T .fwdarw. R transition occurs within the crystal at pH 7 compares very well (rms = 0.19 .ANG. for all atoms) with an R-state structure determined at pH 7 in which the crystals were initially grown in a solution of PAM and malonate at pH 5.9 and subsequently transferred to a buffer containing the ligands at pH 7 (Rpam,mal,crys). In fact, both of the PAM and malonate ligated R-state structures are very similar to both the carbamoyl phosphate and succinate or the N-(phosphonoacetyl)-L-aspartate ligated structures, even though the R-state structures reported here were determined at pH 7. Crystallographic residuals refined to 0.16-0.18 at 2.8-.ANG. resolution for the three structures.