Komives E A, Lougheed J C, Zhang Z, Sugio S, Narayana N, Xuong N H, Petsko G A, Ringe D
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla 92093-0601, USA.
Biochemistry. 1996 Dec 3;35(48):15474-84. doi: 10.1021/bi961556v.
The structural basis for the 3000-fold decrease in catalytic efficiency of the H95N mutant chicken triosephosphate isomerase and the 60-fold regain of catalytic efficiency in the double mutant, H95N.S96P, have been analyzed. The results from a combination of X-ray crystallography and Fourier transform infrared spectroscopy experiments indicate that the predominant defect in the H95N mutant isomerase appears to be its inability to bind the substrate in a coplanar, cis conformation. The structures of each mutant isomerase were determined from X-ray crystallography of the complex of phosphoglycolohydroxamate (PGH), an intermediate analog with the isomerase, and each was solved to a resolution of 1.9 A. The PGH appeared to be in two different conformations in which the enediol-mimicking atoms, O2-N2-C1-O1, of the PGH were not coplanar. No density was observed that would correspond to the coplanar conformation. Two bands are observed for the dihydroxyacetone phosphate carbonyl in the H95N mutant FTIR spectrum, and these can be explained if the O1 of DHAP, like the O1 of PGH in the crystal structure, is in two different positions. Two ordered water molecules are located between O1 of PGH and N delta of N95. Comparison of the structure of the pseudorevertant, H95N.S96P with that for the H95N single mutant, shows that S96P mutation causes the double mutant to regain the ability to bind PGH predominantly in the coplanar, cis conformation. Electron density for a single ordered water molecule bridging the N95 amide side chain and the O2 of PGH is observed, but the density was weak, perhaps indicating that the water molecule is somewhat disordered. Whether or not a water molecule is hydrogen bonded to O2 of PGH may explain the two carbonyl stretching frequencies observed for the GAP carbonyl. Together, the crystal structures and the FTIR data allow a complete explanation of the catalytic properties of these two mutant isomerases.
已对H95N突变型鸡磷酸丙糖异构酶催化效率降低3000倍以及双突变体H95N.S96P催化效率恢复60倍的结构基础进行了分析。X射线晶体学和傅里叶变换红外光谱实验相结合的结果表明，H95N突变型异构酶的主要缺陷似乎是其无法以共面的顺式构象结合底物。通过磷酸甘油异羟肟酸酯（PGH，一种与异构酶的中间类似物）复合物的X射线晶体学确定了每种突变型异构酶的结构，并且每种结构都解析到了1.9 Å的分辨率。PGH似乎处于两种不同的构象中，其中PGH的烯二醇模拟原子O2-N2-C1-O1不共面。未观察到与共面构象相对应的密度。在H95N突变型FTIR光谱中观察到磷酸二羟丙酮羰基有两条谱带，如果磷酸二羟丙酮的O1与晶体结构中PGH的O1一样处于两个不同位置，那么这些谱带就可以得到解释。在PGH的O1和N95的Nδ之间定位了两个有序水分子。假回复突变体H95N.S96P与H95N单突变体的结构比较表明，S96P突变使双突变体重新获得了以共面的顺式构象主要结合PGH的能力。观察到一个单有序水分子桥接N95酰胺侧链和PGH的O2的电子密度，但密度较弱，这可能表明该水分子有点无序。一个水分子是否与PGH的O2形成氢键可以解释甘油醛-3-磷酸羰基观察到的两个羰基伸缩频率。晶体结构和FTIR数据共同对这两种突变型异构酶的催化特性做出了完整解释。
