1H NMR studies on the catalytic subunit of aspartate transcarbamoylase.

The 1H NMR spectrum of the catalytic subunit of Escherichia coli aspartate transcarbamoylase (EC 2.1.3.2) was simplified by using strains auxotrophic for the aromatic amino acids and a growth medium containing fully deuterated Trp, Phe, and His and partially deuterated Tyr. 1H resonances for Tyr in the catalytic trimer (M(r) = 10(5)) were partially resolved into five peaks at 27 degrees C, which above 50 degrees C were further resolved to give a distinct resonance for each of the eight Tyr residues in the polypeptide chain. Experiments on chemically modified catalytic subunits and on a mutant form in which Tyr-165 was converted to Ser-165 led to the assignment of resonances for Tyr-165, Tyr-240, and Tyr-185. Binding of the substrate, carbamoyl phosphate, caused shifts of two of the unassigned resonances, and the subsequent binding of the aspartate analog succinate perturbed the resonances corresponding to Tyr-165 and Tyr-240. The bisubstrate analog N-(phosphonacetyl)-L-aspartate produced a spectrum differing considerably from that caused by the combination of carbamoyl phosphate and succinate. The NMR spectrum for the Tyr-165-->Ser mutant trimer showed clearly that the single amino acid substitution caused conformational changes affecting the environment of residues remote from the position of the replacement. In contrast, the inactive mutant subunit in which Gly-128 was replaced by Asp exhibited a spectrum virtually identical to that of the wild-type protein. However, addition of the substrate carbamoyl phosphate caused a marked change in the spectrum of the mutant enzyme, whereas that of the wild-type trimer was altered only slightly, showing that the effect of the amino acid substitution was manifested in the NMR spectrum only with the liganded enzyme.

大肠杆菌（Escherichia coli）天冬氨酸转氨甲酰酶（aspartate transcarbamoylase，EC 2.1.3.2）催化亚基（catalytic subunit）的1H核磁共振波谱（1H NMR），可借助芳香族氨基酸营养缺陷型菌株，以及含有全氘代色氨酸（Trp）、苯丙氨酸（Phe）、组氨酸（His）与部分氘代酪氨酸（Tyr）的培养基进行简化。在27℃条件下，该催化三聚体（catalytic trimer，相对分子质量M(r)=10^5）中酪氨酸的1H共振峰被部分拆分为5个峰；当温度高于50℃时，这些峰可进一步拆分，使多肽链中8个酪氨酸残基各自产生独立的共振信号。对化学修饰的催化亚基与将Tyr-165突变为Ser-165的突变体（mutant form）开展的实验，完成了对Tyr-165、Tyr-240与Tyr-185共振峰的归属。底物氨甲酰磷酸（carbamoyl phosphate）的结合会引发两个未归属共振峰的化学位移，后续天冬氨酸类似物琥珀酸（succinate）的结合则使对应于Tyr-165和Tyr-240的共振峰发生扰动。双底物类似物（bisubstrate analog）N-(膦酰乙酰基)-L-天冬氨酸（N-(phosphonacetyl)-L-aspartate）所产生的波谱，与氨甲酰磷酸和琥珀酸联合作用所诱导的波谱存在显著差异。Tyr-165→Ser突变三聚体的NMR波谱清晰表明，该单氨基酸替换引发了构象变化，影响了替换位点远端残基的微环境。与之相反，将Gly-128替换为Asp的无活性突变亚基，其波谱与野生型（wild-type）蛋白质几乎完全一致。但加入底物氨甲酰磷酸后，该突变酶的波谱发生了显著变化；而野生型三聚体的波谱仅出现轻微改变，这说明该氨基酸替换的效应仅在结合配体的酶中才会在NMR波谱中得以体现。