CHANGES IN STABILITY AND ALLOSTERIC PROPERTIES OF ASPARTATE TRANSCARBAMOYLASE RESULTING FROM AMINO-ACID SUBSTITUTIONS IN THE ZINC-BINDING DOMAIN OF THE REGULATORY CHAINS
CHANGES IN STABILITY AND ALLOSTERIC PROPERTIES OF ASPARTATE TRANSCARBAMOYLASE RESULTING FROM AMINO-ACID SUBSTITUTIONS IN THE ZINC-BINDING DOMAIN OF THE REGULATORY CHAINS
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DOI：
10.1073/pnas.86.9.3094
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发表时间：
1989-05-01
影响因子：
11.1
通讯作者：
SCHACHMAN, HK
中科院分区：
文献类型：
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作者：
EISENSTEIN, E;MARKBY, DW;SCHACHMAN, HK
来自大肠杆菌的调节酶天冬氨酸转甲氨基酰基酶（ATCase; EC 2.1.3.2）的变抗转变相关的亚基相互作用能的变化部分定位于6个催化(C)和6个调节(r)多肽链之间的界面。位点定向诱变已被用于构建在r链的锌结合域的有限区域进行氨基酸取代的酶。r114 Cys是调节链中结合结构锌离子的四种半胱氨酸之一，用Ser或His取代r114 Cys会导致链折叠不正确，这可以从细胞提取物中无法检测到稳定的组装全酶中看出。在对应的催化三聚体中，r111 Asn在r链和c链之间的界面上被Ala取代，导致野生型ATCase的同向和异向效应完全丧失。此外，沉降速度实验表明，由于相对于R态，T（“紧”）构象优先失稳，在没有活性位点配体的情况下，该突变酶存在于R（“松弛”）构象中。相反，在相邻的r链和c链之间的界面上，Ala取代r113 Asn导致酶的协同性增加。当r139 Lys被Met取代时，Vmax比野生型ATCase降低50%，而当r142 Glu被Asp取代时，Vmax增加约2倍。当全酶与分离的催化亚基孵育时，该区域的氨基酸取代显著影响亚基相互作用能，这是通过亚基交换率来测量的，因此可以测量双底物类似物N-（磷酸乙酰基）- l -天冬氨酸在减弱亚基间相互作用中的作用。r142 Glu .fwdarw的亚基交换增加了约9倍。Asp突变体是r142 Glu .fwdarw突变体的近20倍。Ala在配体存在下发生突变。
Changes in subunit interaction energies linked to the allosteric transition of the regulatory enzyme aspartate transcarbamoylase (ATCase; EC 2.1.3.2) from Escherichia coli are localized in part at interfaces between the six catalytic (C) and six regulatory (r) polypeptide chains. Site-directed mutagenesis has been used to construct enzymes with amino acid substitutions in a limited region of the zinc-binding domain of the r chains. Substitution of Ser or His for r114 Cys, one of four cysteines binding the structural zinc ion in the regulatory chain, leads to incorrectly folded chains as shown by the inability to detect stable assembled holoenzyme in cell extracts. Replacement of r111 Asn by Ala at the interface between an r chain and a c chain in the apposing catalytic trimer causes a complete loss of the homotropic and heterotropic effects characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrated that this mutant enzyme exists in the R ("relaxed") conformation in the absence of active site ligands due to preferential destabilization of the T ("taut") conformation relative to the R state. In contrast, replacement of r113 Asn by Ala at the interface between adjacent r and c chains leads to an increase in the cooperativity of the enzyme. When r139 Lys is replaced by Met, Vmax is reduced by 50% compared to wild-type ATCase, whereas it is increased about 2-fold when r142 Glu is replaced by Asp. Amino acid substitutions in this domain significantly affect subunit interaction energy as measured by rate of subunit exchange when holoenzymes are incubated with isolated catalytic subunits, thus permitting measurements of the effect of the bisubstrate analog N-(phosphonacetyl)-L-aspartate in weakening intersubunit interactions. Subunit exchange increased about 9-fold for the r142 Glu .fwdarw. Asp mutant and almost 20-fold for the r142 Glu .fwdarw. Ala mutant in the presence of the ligand.