STRUCTURE OF RABBIT MUSCLE PYRUVATE-KINASE COMPLEXED WITH MN2+, K+, AND PYRUVATE
STRUCTURE OF RABBIT MUSCLE PYRUVATE-KINASE COMPLEXED WITH MN2+, K+, AND PYRUVATE
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DOI：
10.1021/bi00186a033
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发表时间：
1994-05-24
期刊：
影响因子：
2.9
通讯作者：
REED, GH
中科院分区：
文献类型：
--
作者：
LARSEN, TM;LAUGHLIN, LT;REED, GH
兔肌肉丙酮酸激酶的分子结构，结晶为一个复杂的Mn 2+，K+，和丙酮酸盐，已解决了2.9埃分辨率。研究中使用的晶体属于空间群P1，晶胞尺寸为a = 83.6埃，B = 109.9埃，c = 146.8埃，α = 94.9度，β = 93.6度，γ = 112.3度。不对称单元中有两个四聚体。使用来自猫肌肉的丙酮酸激酶的四聚体的坐标作为搜索模型，通过分子置换解析结构[Muirhead，H.，Claydon，D.一、巴福德，D.，洛里默角G.，福瑟吉尔-吉尔摩湖一、Schiltz，E.，和施密特，W.（1986年）5，475-481]。从编码兔肌肉酶的cDNA衍生的氨基酸序列与电子密度拟合。兔和猫肌肉酶具有相似的94%序列同一性，并且预期折叠模式几乎相同。然而，猫和兔丙酮酸激酶的拓扑模型有三个不同的区域。Mn 2+通过Glu 271和Asp 295的羧酸侧链与蛋白质配位。这两个残基在所有已知的丙酮酸激酶中是严格保守的。此外，Mn 2+的密度与丙酮酸盐的密度有关，这与通过底物的羧酸氧和羰基氧的螯合作用一致。赖氨酸269的ε-NH 2和苏氨酸327的OH位于结合丙酮酸的甲基的两侧。球形电子密度，分配给K+，位于由Thr 113的羰基氧，Ser 76的O-γ，Asn 74的O-δ 1和Asp 112的O-δ 2贡献的四个氧配体的明确定义的口袋内。Asp 112与Lys 269和Arg 72侧链的相互作用可能间接介导单价阳离子对活性的影响。
The molecular structure of rabbit muscle pyruvate kinase, crystallized as a complex with Mn2+, K+, and pyruvate, has been solved to 2.9-Angstrom resolution. Crystals employed in the investigation belonged to the space group P1 and had unit cell dimensions a = 83.6 Angstrom, b = 109.9 Angstrom, c 146.8 Angstrom, alpha = 94.9 degrees, beta = 93.6 degrees, and gamma = 112.3 degrees. There were two tetramers in the asymmetric unit. The structure was solved by molecular replacement, using as the search model the coordinates of the tetramer of pyruvate kinase from cat muscle [Muirhead, H., Claydon, D. A., Barford, D., Lorimer, C. G., Fothergill-Gilmore, L. A., Schiltz, E., and Schmitt, W. (1986) EMBOJ. 5, 475-481]. The amino acid sequence derived from the cDNA coding for the enzyme from rabbit muscle was fit to the electron density. The rabbit and cat muscle enzymes have similar to 94% sequence identity, and the folding patterns are expected to be nearly identical. There are, however, three regions where the topological models of the cat and rabbit pyruvate kinases differ. Mn2+ coordinates to the protein through the carboxylate side chains of Glu 271 and Asp 295. These two residues are strictly conserved in all known pyruvate kinases. In addition, the density for Mn2+ is connected to that of pyruvate, consistent with chelation through a carboxylate oxygen and the carbonyl oxygen of the substrate. The epsilon-NH2 of Lys 269 and the OH of Thr 327 lie on either side of the methyl group of bound pyruvate. Spherical electron density, assigned to K+, is located within a well-defined pocket of four oxygen ligands contributed by the carbonyl oxygen of Thr 113, O-gamma of Ser 76, O-delta 1 of Asn 74, and O-delta 2 of Asp 112. The interaction of Asp 112 with the side chains of Lys 269 and Arg 72 may mediate, indirectly, monovalent cation effects on activity.