Preparation of enantiomerically pure L-7-azatryptophan by an enzymatic method and its application to the development of a fluorimetric activity assay for tryptophanyl-tRNA synthetase.
Preparation of enantiomerically pure L-7-azatryptophan by an enzymatic method and its application to the development of a fluorimetric activity assay for tryptophanyl-tRNA synthetase.
复制标题
通过酶法制备对映体纯的 L-7-氮杂色氨酸及其在色氨酸-tRNA 合成酶荧光活性测定中的应用。
作者：
J. Brennan;C. Hogue;B. Rajendran;K. Willis;A. Szabo
在无机焦磷酸酶存在下，D，L-7-氮杂色氨酸（D，L-7AW）与三羟甲基-tRNA合成酶（TrpRS）、三磷酸腺苷（ATP）和Mg 2+的反应导致形成高度荧光的1 - 7AW-腺苷酸复合物。该复合物的检测是基于其在加入ATP后在315 nm激发和360 nm发射处增强的荧光。该立体选择性反应用于使用市售外消旋D，L-7AW开发TrpRS的活性测定。该测定法可用于测定250微升样品中含有少于1 nmol酶的样品中TrpRS的活性。因此，可以评估酶活性，而不求助于tRNATrp酰化的放射性测定。立体选择性测定的第二用途是确认纯L-7AW、D-7AW或两种对映异构体的混合物的存在。通过使D，L-7AW与氯乙酸酐反应以形成N-氯乙酰基-D，L-7AW（ClAc-7AW），随后使用羧肽酶A立体特异性蛋白水解消化ClAc-7AW以产生游离L-7AW来制备D-7AW和L-7AW。反相高效液相色谱法可将L-7AW与未反应的N-氯乙酰基-7AW分离。基于TrpRS的测定能够明确区分7AW的两种对映异构体。然后使用该测定法来鉴定在三肽L-赖氨酰-D，L-7-氮杂色氨酸-L-赖氨酸的解析级分中存在7AW的哪种对映异构体。用蛋白酶消化解析的三肽产生7AW的游离L或D对映体，其使用TrpRS测定程序容易地鉴定。
The reaction of D,L-7-azatryptophan (D,L-7AW) with tryptophanyl-tRNA synthetase (TrpRS), adenosine triphosphate (ATP), and Mg2+ in the presence of inorganic pyrophosphatase results in the formation of a highly fluorescent l-7AW-adenylate complex. Detection of this complex is based on its enhanced fluorescence at 315 nm excitation and 360 nm emission after the addition of ATP. This stereoselective reaction was used to develop an activity assay for TrpRS using commercially available racemic D,L-7AW. The assay can be used to determine the activity of TrpRS from samples which contain less than 1 nmol of enzyme in 250 microL of sample. Thus the enzyme activity can be assessed without resorting to a radioactive assay of tRNATrp acylation. A secondary use of the stereoselective assay was for confirming the presence of pure L-7AW, D-7AW, or mixtures of the two enantiomers. D-7AW and L-7AW were prepared by reacting D,L-7AW with chloroacetic anhydride to form N-chloroacetyl-D,L-7AW (ClAc-7AW) followed by stereospecific proteolytic digestion of ClAc-7AW using carboxypeptidase A to produce the free L-7AW. The L-7AW could be separated from unreacted N-chloroacetyl-7AW by reverse-phase HPLC. The TrpRS-based assay was able to unambiguously discriminate between the two enantiomers of 7AW. The assay was then used to identify which enantiomer of 7AW was present in resolved fractions of the tripeptide L-lysyl-D,L-7-azatryptophyl-L-lysine. Digestion of the resolved tripeptides with protease enzymes produced the free L or D enantiomer of 7AW, which was easily identified using the TrpRS assay procedure.