Peterson C B, Schachman H K
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Biol Chem. 1992 Feb 5;267(4):2443-50.
A single alpha-helical polypeptide segment of 21 amino acids near the carboxyl terminus of the catalytic chain of aspartate transcarbamoylase from Escherichia coli has been shown recently to be important for the in vivo folding of the chains and assembly of the enzyme (Peterson, C. B., and Schachman, H. K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 458-462). Calorimetric measurements on purified mutant enzymes showed that single amino acid replacements within this secondary structural element affect the overall thermal stability of the oligomeric enzyme and the energetics of the interactions between polypeptide chains within the holoenzyme. Studies presented here demonstrate that marked changes in cooperativity occur due to single amino acid substitutions. Replacement of Gln288 by either Ala or Glu leads to a striking increase in the Hill coefficient of the holoenzymes and a substantial increase in the aspartate concentration corresponding to one-half Vmax. In contrast, the isolated catalytic trimers harboring these same substitutions were similar in activity to the wild-type subunit, with the same affinity for aspartate as indicated by the values of Km. Substituting Ala for the only charged residue in the helix, Arg296, caused a marked reduction in enzyme activity, as well as a greatly reduced stability of the holoenzyme due to a substantial weakening of the interactions between the catalytic and regulatory subunits. A subunit exchange method was used to demonstrate the changes in interchain interactions resulting from the amino acid substitutions and to show the additional weakening upon the binding of the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate, at the active sites. Taken together, the results on this series of mutant enzymes illustrate how the effects of single amino acid replacements in one element of secondary structure are propagated throughout the molecule to positions remote from the site of the substitution.
最近的研究表明，大肠杆菌天冬氨酸转氨甲酰酶催化链羧基末端附近一个由21个氨基酸组成的单α-螺旋多肽片段，对该酶链的体内折叠和组装非常重要（彼得森，C.B.，和沙克曼，H.K.（1991年）《美国国家科学院院刊》88，458 - 462）。对纯化的突变酶进行的量热测量表明，该二级结构元件内的单个氨基酸替换会影响寡聚酶的整体热稳定性以及全酶内多肽链之间相互作用的能量学。本文所呈现的研究表明，单个氨基酸替换会导致协同性发生显著变化。用丙氨酸或谷氨酸替换Gln288会导致全酶的希尔系数显著增加，并且对应于一半Vmax的天冬氨酸浓度大幅增加。相反，带有这些相同替换的分离催化三聚体在活性上与野生型亚基相似，Km值表明它们对天冬氨酸的亲和力相同。用丙氨酸替换螺旋中唯一的带电荷残基Arg296会导致酶活性显著降低，同时由于催化亚基和调节亚基之间相互作用的大幅减弱，全酶的稳定性也大大降低。采用亚基交换方法来证明氨基酸替换导致的链间相互作用变化，并展示在活性位点结合双底物配体N -（膦酰乙酰）-L -天冬氨酸后相互作用的进一步减弱。综上所述，这一系列突变酶的结果说明了二级结构中一个元件内单个氨基酸替换的影响是如何在整个分子中传播到远离替换位点的位置的。
