The ionization states of the 5'-phosphate group in the various coenzyme forms bound to mitochondrial aspartate aminotransferase.
The ionization states of the 5'-phosphate group in the various coenzyme forms bound to mitochondrial aspartate aminotransferase.
复制标题
与线粒体天冬氨酸转氨酶结合的各种辅酶形式中 5-磷酸基团的电离状态。
DOI：
10.1016/0003-9861(91)90006-5
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发表时间：
1991
影响因子：
3.9
通讯作者：
Martinez-Carrion,M
中科院分区：
文献类型：
--
作者：
Sanchez-Ruiz,JM;Iriarte,A;Martinez-Carrion,M
我们已经进行了傅立叶变换红外光谱研究线粒体天冬氨酸氨基转移酶的光谱区域，磷酸单酯引起的吸收。上述区域的红外光谱主要由蛋白质吸收决定。然而，低于1020 cm− 1的蛋白质干扰较小，允许检测由双阴离子磷酸单酯的对称拉伸产生的条带[T. Shimanouchi，M. Tsuboi和Y. Kyogoku（1964）Adv.Chem.Phys.8，435-498]。几种酶形式（磷酸吡哆醛、磷酸吡哆胺和硼氢化钠还原的磷酸吡哆醛形式）中该条带的积分强度不随pH值在5-9范围内变化。该行为与溶液中游离磷酸吡哆醛（PLP）和磷酸吡哆胺（PMP）的行为形成对比，其中相同红外带强度与pH的依赖性可与溶液中5′-磷酸酯的已知pK值相关。在用硼氢化钠还原之前和之后，PLP酶形式的该红外带的积分强度值接近于由游离PLP在pH 8-9下给出的积分强度值。这些结果表明，在线粒体天冬氨酸转氨酶的活性部位，PLP的5′-磷酸基团即使在pH值接近5时也主要保持双阴离子。因此，这表明，化学位移的变化与pH滴定的各种形式的PLP在以前的31 P NMR研究这种酶的报告[M。E.马丁利，J. R。马丁利和M. Martinez-Carrion（1982）J.Biol.Chem.257，8872]是由于磷化学位移感测到由附近残基的电离引起的O → P → O键畸变。由于没有化学位移的变化，在pH滴定的PMP形式（缺乏一个电离的内部醛亚胺）的这种同工酶，在活性位点的PLP和赖氨酸-258之间的席夫碱是最有可能的候选人的电离基团影响磷化学位移在这种酶。
We have carried out a Fourier transform infrared spectroscopic study of mitochondrial aspartate aminotransferase in the spectral region where phosphate monoesters give rise to absorption. Infrared spectra in the above-mentioned region are dominated by protein absorption. Yet, below 1020 cm−1protein interferences are minor, permitting the detection of the band arising from the symmetric stretching of dianionic phosphate monoesters [T. Shimanouchi, M. Tsuboi, and Y. Kyogoku (1964)Adv. Chem. Phys.8, 435–498]. The integrated intensity of this band in several enzyme forms (pyridoxal phosphate, pyridoxamine phosphate, and sodium borohydride-reduced, pyridoxyl phosphate form) does not change with pH in the range 5–9. This behavior contrasts that of free pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP) in solution, where the dependence of the same infrared band intensity with pH can be correlated to the known pKvalues for the 5′-phosphate ester in solution. The integrated intensity value of this infrared band for the PLP enzyme form before and after reduction with sodium borohydride is close to that given by free PLP at pH 8–9. These results are taken as evidence that in the active site of mitochondrial aspartate aminotransferase the 5′-phosphate group of PLP remains mostly dianionic even at a pH near 5. Thus, it is suggested that the chemical shift changes associated with pH titrations of various PLP forms reported in a previous31P NMR study of this enzyme [M. E. Mattingly, J. R. Mattingly, and M. Martinez-Carrion (1982)J. Biol. Chem.257, 8872] are due to the fact that the phosphorus chemical shift senses the OPO bond distortions induced by the ionization of a nearby residue. Since no chemical shift changes were observed in pH titrations of the PMP forms (lacking an ionizable internal aldimine) of this isozyme, the Schiff base between PLP and Lys-258 at the active site is the most likely candidate for the ionizing group influencing the phosphorus chemical shift in this enzyme.