Sanchez-Ruiz J M, Iriarte A, Martinez-Carrion M
Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri-Kansas City 64110-2499.
Arch Biochem Biophys. 1991 Apr;286(1):38-45. doi: 10.1016/0003-9861(91)90006-5.
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-1 protein 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 pK values 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 previous 31P 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 O-P-O 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.
我们对线粒体天冬氨酸氨基转移酶进行了傅里叶变换红外光谱研究，研究区域为磷酸单酯产生吸收的光谱区域。上述区域的红外光谱以蛋白质吸收为主。然而，在1020cm-1以下，蛋白质干扰较小，使得可以检测到由二价阴离子磷酸单酯的对称伸缩产生的谱带[T. 岛野内、M. 津部井和Y. 京极（1964年）《化学物理进展》8，435 - 498]。该谱带在几种酶形式（磷酸吡哆醛、磷酸吡哆胺以及硼氢化钠还原的磷酸吡哆醛形式）中的积分强度在pH值5 - 9范围内不随pH变化。这种行为与溶液中游离的磷酸吡哆醛（PLP）和磷酸吡哆胺（PMP）不同，在溶液中相同红外谱带强度对pH的依赖性与溶液中5'-磷酸酯的已知pK值相关。硼氢化钠还原前后PLP酶形式的该红外谱带积分强度值接近pH 8 - 9时游离PLP给出的值。这些结果被视为证据，表明在线粒体天冬氨酸氨基转移酶的活性位点，即使在pH接近5时，PLP的5'-磷酸基团大多仍为二价阴离子。因此，有人提出，在先前对该酶的31P NMR研究[M. E. 马丁利、J. R. 马丁利和M. 马丁内斯 - 卡里翁（1982年）《生物化学杂志》257，8872]中报道的各种PLP形式的pH滴定相关化学位移变化，是由于磷化学位移感知到附近残基电离引起的O - P - O键扭曲。由于在该同工酶的PMP形式（缺乏可电离的内部醛亚胺）的pH滴定中未观察到化学位移变化，活性位点处PLP与Lys - 258之间的席夫碱是影响该酶中磷化学位移的最可能的电离基团候选者。
