CRYSTAL-STRUCTURE OF THE COMPLEX OF CARBOXYPEPTIDASE-A WITH A STRONGLY BOUND PHOSPHONATE IN A NEW CRYSTALLINE FORM - COMPARISON WITH STRUCTURES OF OTHER COMPLEXES
CRYSTAL-STRUCTURE OF THE COMPLEX OF CARBOXYPEPTIDASE-A WITH A STRONGLY BOUND PHOSPHONATE IN A NEW CRYSTALLINE FORM - COMPARISON WITH STRUCTURES OF OTHER COMPLEXES
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DOI：
10.1021/bi00475a019
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发表时间：
1990-06-12
期刊：
影响因子：
2.9
通讯作者：
LIPSCOMB, WN
中科院分区：
文献类型：
--
作者：
KIM, H;LIPSCOMB, WN
O-[[（1 R）-[[N-（苯基甲氧碳）-L-丙氨酰]氨基]乙基]羟基氧膦基]-L-3-苯基乙酸酯[ZAAP（O）F]是（苄氧羰基）-Ala-Ala-Phe或（苄氧羰基）-Ala-Ala-苯基乳酸酯的类似物，它与羧肽酶A有很强的亲和力（Ki = 3 pM）。类似的膦酸酯已被证明是CPA催化水解的过渡态类似物[Hanson，J.E.，Kaplan，A.P.，和Bartlett，P.A.（1989）Biochemistry 28，6294-6305]。在本研究中，这种膦酸酯与羧肽酶A的复合物的结构已通过X-射线晶体学测定到2.0埃的分辨率。该复合物在空间群P212121中结晶，晶胞尺寸为α。= 61.9埃，B = 67.2埃，和c = 76.2埃。配合物的结构通过分子置换得到解析。针对10.0和2.0埃之间的20776个独特反射的结构的精细化。产生0.193的晶体学残差，包括140个水分子。抑制剂的两个氧膦基氧在2.2埃处与活性部位锌结合。在亲电（Arg-127）侧和3.1.在亲核（Glu-270）侧。这种膦酸盐抑制剂的结合模式的各种功能是一致的假设，羧肽酶A催化的水解进行通过一般碱的机制，其中底物的羰基碳被锌-羟基（或锌-水）攻击。结合的抑制剂的一个意想不到的特征，即与丙氨酸连接的苄氧基羰基处的顺式氨基甲酰基酯键，允许抑制剂的苄氧基羰基苯环与Tyr-198有利地相互作用。这种复杂的结构与以前的结构相比，羧肽酶A，包括复合物与马铃薯抑制剂，水合酮亚甲基底物类似物，和磷酰胺抑制剂。还与嗜热菌蛋白酶与一些膦酰胺抑制剂的复合物进行了比较。
O-[[(1R)-[[N-(Phenylmethoxycarbon)-L-alanyl]amino]ethyl]hydroxyphosphinyl]-L-3-phenyllacetate [ZAAP(O)F], an analogue of (benzyloxycarbonyl)-Ala-Ala-Phe or (benzyloxycarbonyl)-Ala-Ala-phenyllactate, binds to carboxypeptidase A with great affinity (Ki = 3 pM). Similar phosphonates have been shown to be transition-state analogues of the CPA-catalyzed hydrolysis [Hanson, J.E., Kaplan, A.P., and Bartlett, P.A. (1989) Biochemistry 28, 6294-6305]. In the present study, the structure of the complex of this phosphonate with carboxypeptidase A has been determined by X-ray crystallography to a resolution of 2.0 .ANG.. The complex crystallizes in the space group P212121 with cell dimensions .alpha. = 61.9 .ANG., b = 67.2 .ANG., and c = 76.2 .ANG.. The structure of the complex was solved by molecular replacement. Refinement of the structure against 20776 unique reflections between 10.0 and 2.0 .ANG. yields a crystallographic residual of 0.193, including 140 water molecules. The two phosphinyl oxygens of the inhibitor bind to the active-site zinc at 2.2 .ANG. on the electrophilic (Arg-127) side and 3.1 .ANG. on the nucleophilic (Glu-270) side. Various features of the binding mode of this phosphonate inhibitor are consistent with the hypothesis that carboxypeptidase A catalyzed hydrolysis proceeds through a general-base mechanism in which the carbonyl carbon of the substrate is attacked by Zn-hydroxyl (or Zn-water). An unexpected feature of the bound inhibitor, the cis carbamoyl ester bond at the benzyloxycarbonyl linkage to alanine, allows the benzyloxycarbonyl phenyl ring of the inhibitor to interact favorably with Tyr-198. This complex structure is compared with previous structures of carboxypeptidase A, including the complexes with the potato inhibitor, a hydrated keto methylene substrate analogue, and a phosphonamidate inhibitor. Comparisons are also made with the complexes of thermolysin with some phosphonamidate inhibitors.